U.S. patent application number 13/508181 was filed with the patent office on 2012-09-06 for immune system modulating composition.
Invention is credited to Jose Antonio Vazquez Boland.
Application Number | 20120225078 13/508181 |
Document ID | / |
Family ID | 41509193 |
Filed Date | 2012-09-06 |
United States Patent
Application |
20120225078 |
Kind Code |
A1 |
Vazquez Boland; Jose
Antonio |
September 6, 2012 |
IMMUNE SYSTEM MODULATING COMPOSITION
Abstract
Rhodococcus equi (R.equi) has been determined to have a major
adhesion factor encoded by a rpl pathogenicity island which enables
host colonisation, wherein the rpl pathogenicity islandis absent
from non-pathogenic Rhodococcus species. Further, the proteins
(Rpl) encoded by the rpl pathogenicity island have been determined
to be major immunodominant antigens. There is provided a novel
diagnostic marker and vaccine candidate for R. equi in horses and
other susceptible species.
Inventors: |
Vazquez Boland; Jose Antonio;
(Edinburgh, GB) |
Family ID: |
41509193 |
Appl. No.: |
13/508181 |
Filed: |
November 11, 2010 |
PCT Filed: |
November 11, 2010 |
PCT NO: |
PCT/GB2010/051889 |
371 Date: |
May 4, 2012 |
Current U.S.
Class: |
424/139.1 ;
424/190.1; 424/245.1; 435/320.1; 435/325; 435/6.12; 435/7.92;
436/501; 506/9; 514/1.1; 514/44R; 530/324; 530/326; 530/387.2;
530/387.9; 536/23.7 |
Current CPC
Class: |
C12Q 1/689 20130101;
C07K 14/36 20130101; C07K 2317/76 20130101; G01N 2333/34 20130101;
G01N 33/56911 20130101; A61K 39/05 20130101; C07K 16/1267 20130101;
A61K 2039/552 20130101; A61K 2039/53 20130101; G01N 2500/04
20130101 |
Class at
Publication: |
424/139.1 ;
424/190.1; 424/245.1; 435/6.12; 435/7.92; 435/325; 435/320.1;
436/501; 506/9; 514/1.1; 514/44.R; 530/324; 530/326; 530/387.2;
530/387.9; 536/23.7 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/68 20060101 C12Q001/68; G01N 33/566 20060101
G01N033/566; C12N 5/10 20060101 C12N005/10; C12N 15/63 20060101
C12N015/63; C07H 21/04 20060101 C07H021/04; A61K 38/00 20060101
A61K038/00; A61K 31/7088 20060101 A61K031/7088; C07K 14/34 20060101
C07K014/34; C07K 7/08 20060101 C07K007/08; C07K 16/42 20060101
C07K016/42; C07K 16/12 20060101 C07K016/12; A61K 39/00 20060101
A61K039/00; C40B 30/04 20060101 C40B030/04 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 11, 2009 |
GB |
0919733.6 |
Claims
1. A polypeptide associated with pilus formation in R. equi
comprising an amino acid sequence encoded by a polynucleotide
sequence as set forth in any one of SEQ ID NO 1, SEQ ID NO 2, SEQ
ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID
NO 8, and SEQ ID NO 9 or a fragment, derivative or variant of such
a polypeptide.
2. A polypeptide as claimed in claim 1 comprising an amino acid
sequence encoded by a polynucleotide sequence as set forth in any
one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID
NO 5, SEQ ID NO 8, and SEQ ID NO 9 or a fragment, derivative or
variant of such a polypeptide.
3. A polypeptide sequence as claimed in claim 1 comprising an amino
acid sequence encoded by a polynucleotide sequence as set forth in
any one of SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4 or a fragment,
derivative or variant of such a polypeptide.
4. A polypeptide sequence as claimed in claim 1 wherein the
polypeptide is encoded by a polynucleotide sequence comprising
ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA
GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC
GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT
CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG
ACGATCCCGGCGGCGAGTAG (SEQ ID NO 2) or a fragment, derivative or
variant of such a polypeptide.
5. A polypeptide as claimed in claim 1 wherein the polypeptide
comprises an amino acid sequence
MNLFFANLYLMGLDVKDRLTRDDRGATAVEYGLMVAGIAMVIIVAVFAFG
DKITDLFDGFNFDDPGGE (SEQ ID NO 10) or a fragment, derivative or
variant of such a polypeptide.
6. A polypeptide as claimed in claim 1 wherein the polypeptide
comprises an amino acid sequence DKITDLFDGFNFDDPGGE (SEQ ID NO 11)
or a fragment, or derivative or variant of such a polypeptide.
7. A polypeptide as claimed in claim 1 wherein the polypeptide
comprises an amino acid sequence DKITDLFDGFNFDDPGGE (SEQ ID NO 11)
or a fragment, or derivative of such a polypeptide.
8. A polypeptide as claimed in claim 1 wherein the polypeptide
comprises an amino acid sequence DKITDLFDGFNFDDPGGE (SEQ ID NO
11).
9. A composition comprising a polypeptide or a fragment,
derivative, or variant thereof according to claim 1, together with
a pharmaceutically acceptable carrier.
10. An antibody or an antigen binding fragment of said antibody
which has binding specificity to a polypeptide according to claim
1.
11. An anti-idiotypic antibody which has binding specificity to an
antibody or an antigen binding fragment of said antibody of claim
10.
12. A construct comprising an isolated nucleic acid sequence which
encodes a polypeptide as claimed in claim 1 operably linked to a
promoter which is functional to allow transcription of the nucleic
acid sequence.
13. At least (i) one polypeptide associated with pilus formation in
R. equi or a fragment, derivative or variant thereof, or (ii) one
nucleic acid which encodes least one polypeptide associated with
pilus formation in R. equi, or (iii) one antibody or an antigen
binding fragment of said antibody which has binding specificity to
at least one polypeptide associated with pilus formation in R.
equi, or (iv) one construct comprising an isolated nucleic acid
molecule which encodes a polypeptide as claimed in claim 1 operably
linked to a promoter which is functional to allow transcription of
the nucleic acid sequence for use in medicine.
14. At least (i) one polypeptide comprising an amino acid sequence
encoded by a polynucleotide sequence selected from any one of SEQ
ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID
NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a fragment,
derivative, or variant thereof as claimed in claim 1, or (ii) one
antibody or an antigen binding fragment of said antibody which has
binding specificity to such a polypeptide, or (iii) one construct
comprising an isolated nucleic acid molecule which encodes such a
polypeptide as claimed in claim 1 operably linked to a promoter
which is functional to allow transcription of the nucleic acid
sequence for use in medicine, preferably in the treatment or
prevention of a disease caused by R. equi.
15. A polypeptide comprising an amino acid sequence encoded by a
polynucleotide sequence selected from any one of SEQ ID NO 1, SEQ
ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID
NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a fragment, derivative, or
variant thereof as claimed in claim 1 for use in the treatment or
prevention of a disease caused by R. equi.
16. An antibody or an antigen binding fragment of said antibody
which has binding specificity to a polypeptide comprising an amino
acid sequence encoded by a polynucleotide sequence selected from
any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ
ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 or
a fragment, derivative, or variant thereof for use in the treatment
or prevention of a disease caused by R. equi.
17. An isolated nucleic acid molecule which encodes a polypeptide
comprising an amino acid sequence encoded by a polynucleotide
sequence selected from any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID
NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO
8, and SEQ ID NO 9 or a fragment, derivative, or variant thereof
for use in the treatment or prevention of a disease caused by R.
equi.
18. A construct comprising an isolated nucleic acid molecule which
encodes a polypeptide comprising an amino acid sequence encoded by
a polynucleotide sequence selected from any one of SEQ ID NO 1, SEQ
ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID
NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a fragment, derivative, or
variant thereof operably linked to a promoter which is functional
to allow transcription of the nucleic acid sequence for use in the
treatment or prevention of a disease caused by R. equi.
19. A method of treating or preventing a disease or condition, in
particular a disease or condition caused by R. equi, comprising the
step of administering (i) at least one polypeptide associated with
pilus formation in R. equi, (ii) a nucleic acid which encodes least
one polypeptide associated with pilus formation in R. equi, or
(iii) an antibody or an antigen binding fragment of said antibody
which has binding specificity to at least one polypeptide
associated with pilus formation in R. equi, to a subject, in
particular a subject suffering from, or suspected to be suffering
from, or at risk of a condition mediated by R. equi.
20. The method as claimed in claim 19 comprising the step of
administering (i) a polypeptide comprising an amino acid sequence
encoded by a polynucleotide sequence selected from any one of SEQ
ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID
NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a fragment,
derivative, or variant thereof as claimed in claim 1, or (ii) an
antibody or an antigen binding fragment of said antibody which has
binding specificity to such a polypeptide, or (iii) a construct
comprising an isolated nucleic acid molecule which encodes such a
polypeptide as claimed in claim 1 operably linked to a promoter
which is functional to allow transcription of the nucleic acid
sequence to a subject, in particular a subject suffering from, or
suspected to be suffering from, or at risk of a condition mediated
by R. equi.
21. A method of detecting R. equi in a sample comprising the step
of detecting (i) a polypeptide comprising an amino acid sequence
encoded by a polynucleotide sequence selected from any one of SEQ
ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID
NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a fragment,
derivative, or variant thereof as claimed in claim 1, or (ii) an
antibody or an antigen binding fragment of said antibody which has
binding specificity to such a polypeptide, or (ii) a nucleic acid
molecule which encodes such a polypeptide as claimed in claim 1 in
a sample.
22. The method as claimed in claim 21 for diagnosing a disease or
condition caused by R. equi comprising the step of detecting (i) a
polypeptide associated with Rpl pilus formation, preferably an
amino acid sequence encoded by a polynucleotide sequence selected
from any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4,
SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9
or a fragment, derivative, or variant thereof as claimed in claim
1, or (ii) an antibody or an antigen binding fragment of said
antibody which has binding specificity to such a polypeptide, or
(ii) a nucleic acid molecule which encodes such a polypeptide as
claimed in claim 1 in a biological sample from a subject suffering
from, suspected to be suffering from, or at risk of such a
condition.
23. A kit for use in the method of detecting R. equi wherein the
kit comprises (i) a polypeptide associated with Rpl pilus
formation, preferably comprising an amino acid sequence encoded by
a polynucleotide sequence selected from any one of SEQ ID NO 1, SEQ
ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID
NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a fragment, derivative, or
variant thereof as claimed in claim 1, or (ii) an antibody or an
antigen binding fragment of said antibody which has binding
specificity to such a polypeptide, or (iii) nucleic acid probes
capable of binding to a nucleic acid sequence which encodes a
polypeptide associated with pilus formation, preferably at least
one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID
NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9, under
stringent conditions.
24. A kit for use in the method of claim 22 wherein the kit
comprises (i) a polypeptide comprising an amino acid sequence
encoded by a polynucleotide sequence selected from any one of SEQ
ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID
NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a fragment,
derivative, or variant thereof as claimed in claim 1, or (ii) an
antibody or an antigen binding fragment of said antibody which has
binding specificity to such a polypeptide, or (iii) nucleic acid
probes capable of binding to a nucleic acid sequence which encodes
a polypeptide associated with pilus formation, preferably at least
one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID
NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9, under
stringent conditions.
25. A method of screening for agents capable of binding to a
polypeptide comprising an amino acid sequence encoded by a
polynucleotide sequence selected from any one of SEQ ID NO 1, SEQ
ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID
NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a fragment, derivative, or
variant thereof as claimed in claim 1 comprising the steps:
providing a candidate immunogenic R. equi polypeptide comprising an
amino acid sequence encoded by a polynucleotide sequence selected
from any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4,
SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9
or a fragment, derivative, or variant thereof as claimed in claim
1, providing a test agent to the candidate immunogenic R. equi
polypeptide, and determining whether said test agent can bind to
said candidate immunogenic R. equi polypeptide.
26. An isolated or recombinant nucleic acid encoding a polypeptide
associated with pilus formation in R. equi.
27. An isolated or recombinant nucleic acid as claimed in claim 26
comprising a polynucleotide sequence comprising a sequence as set
forth in any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID
NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ
ID NO 9.
28. An isolated or recombinant nucleic acid as claimed in claim 26
comprising a polynucleotide sequence comprising a sequence as set
forth in any one of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID
NO 4, SEQ ID NO 5, SEQ ID NO 8, and SEQ ID NO 9.
29. An isolated or recombinant nucleic acid as claimed in claim 26
comprising a polynucleotide sequence comprising a sequence as set
forth in any one of SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4.
30. An isolated or recombinant nucleic acid as claimed in claim 26
comprising a polynucleotide sequence comprising a sequence as set
forth by SEQ ID NO 2.
31. An isolated or recombinant nucleic acid as claimed in claim 26
comprising a polynucleotide sequence consisting of a sequence as
set forth by SEQ ID NO 2.
32. A vector comprising an isolated or recombinant nucleic acid as
claimed in claim 26.
33. An isolated or recombinant cell comprising a vector as claimed
in claim 32.
34. A composition capable of generating an immune response in a
host comprising one or more surface-associated or secreted
polypeptides of R. equi wherein said polypeptides are associated
with formation of pili of R. equi.
35. A composition as claimed in claim 34 wherein said composition
comprises (i) at least one polypeptide associated with pilus
formation in R. equi, (ii) a nucleic acid which encodes least one
polypeptide associated with pilus formation in R. equi, or (iii) a
polypeptide comprising an amino acid sequence encoded by a
polynucleotide sequence selected from any one of SEQ ID NO 1, SEQ
ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID
NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a fragment, derivative, or
variant thereof as claimed in claim 1, or (iv) a construct
comprising an isolated nucleic acid molecule which encodes such a
polypeptide as claimed in claim 1 operably linked to a promoter
which is functional to allow transcription of the nucleic acid
sequence.
36. Use of the composition of claim 34 to vaccinate a subject such
that R. equi infection in the subject is inhibited or
minimised.
37. Use as claimed in claim 36 wherein the subject is a horse or a
foal.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to polypeptides encoded by
Rhodococcus (Corynebacterium) equi (R. equi), compositions
including such polypeptides (Rpl) and antibodies to such
polypeptides, which can be useful in the treatment of animals,
specifically horses and foals, to minimise infection of animals, by
R. equi. The invention further relates to methods of detection of
R. equi using polypeptides (Rpl), antibodies with binding
specificity to said polypeptides or nucleic acids or the like with
binding specificity to nucleic acids encoding such polypeptides
using, for example, PCR.
BACKGROUND TO THE INVENTION
[0002] Rhodococcus equi is a Gram-positive, facultative
intracellular coccobacillus classified in the order of
Acitnomycetales. It is primarily a soil organism. It has been
recognised as a positive agent of a debilitating and potentially
fatal bronchopneumonia affecting foals worldwide. R. equi is
considered to be one of the most significant pathogens in the
equine breeding industry.
[0003] The successful early diagnosis and treatment of Rhodococcus
equi in foals and management of the foals environment to reduce the
risk of contracting the disease are, arguably, among the most
challenging experiences currently facing equine stud farms.
Presently the treatment of R. equi disease is by the prolonged
administration of a combination of antimicrobials, macrolides, i.e.
erythromycin, azithromycin or clarithromycin, and rifampicin.
However, as this therapy risks antibiotic resistance and adverse
drug reactions in the foal and the dam, improved means of therapy
and prophylactic treatment are required.
[0004] R. equi can also affect non-equine species. In pigs R. equi
is associated with granulomatous lymphadenitis of cervical
lymphatic tissue and in man R. equi can cause cavitary pneumonia,
predominantly in immunocompromised individuals especially those
with acquired immune deficiency syndrome (AIDS). As a consequence
of the AIDS pandemic, R. equi pneumonia has become a disease of
increasing significance in human medicine. R. equi infections have
also been described in cattle, sheep, goats, lama, cats and dogs,
but disease in these species is rare with lesions confined to lymph
node abscessation or wound infection.
[0005] Infection by R. equi relies on the ability of R. equi to
colonise the airways and replicate inside macrophages which is
dependent on its capacity to interfere with endosomal maturation
following phagocytosis and to prevent acidification of the vacuole
in which it resides. Eventually, intracellular proliferation of the
pathogen leads to the necrotic death of the marcophages accompanied
by massive damage to lung tissue characterised by cavitation and
granuloma formation.
[0006] Studies of the virulent strains of R. equi have determined
that such strains possess an extra chromosomal DNA element known as
a plasmid, which is associated with virulence. Plasmids isolated
from regular strains infecting foals have been proposed to include
a region that represents a pathogenicity island, which is a DNA
fragment containing genes required for virulence. The pathogenicity
island identified contains a family of nine virulence associated
protein (Vap) chains (VapA-VapC-Vap-I, pseudo-VapE).
Killed/inactivated R. equi organisms do not illicit protective
immunity, and there is no consistent evidence that protein or DNA
vaccines, based on the highly immunogenic VapA surface antigen, are
efficacious in producing protection against a Rhodococcal pneumonia
in foals. In view of the lack of an efficacious vaccine, R. equi
infection is a major cause of mortality in young foals and the
heavy economic losses incurred due to R. equi has a major economic
impact in countries where thoroughbred racing and breeding is
important (USA, Australia, Ireland, Argentina, UK, France, Spain,
Germany, Austria, Japan etc.). There is a need for treatment
regimes and a vaccine to be developed which can be used to control
R. equi on farms, in particular stud farms.
SUMMARY OF THE INVENTION
[0007] The inventors have determined a novel diagnostic marker and
vaccine candidate for Rhodococcus equi in horses and other
susceptible species and treatment means. Specifically, the
inventors have identified a rpl pathogenicity island that differs
from the yap pathogenicity island and the inventors have determined
the rpl pathogenicity island, in particular RplB, encodes a major
adhesion factor of R. equi which enables host colonisation. The
proteins (Rpl) encoded by the rpl pathogenicity island are
considered to be major immunodominant antigens. The inventors have
further determined that the rpl pathogenicity island is absent from
non-pathogenic Rhodococcus species. These findings allow the use of
probes to proteins or nucleic acid of the rpl pathogenicity island
and antibodies with binding specificity to the proteins encoded by
the rpl pathogenicity island in methods of detection of R. equi.
Further, it enables the use of nucleic acids encoding proteins or
proteins of the rpl pathogenicity island as immune system
modulators, in particular to provoke a protective immune response
to subsequent antigen challenge in an animal.
[0008] Accordingly, a first aspect of the invention provides at
least one immunogenic R. equi polypeptide having an amino acid
sequence, encoded by a polynucleotide sequence comprising a
polynucleotide sequence of a gene selected from a gene as listed at
table one, or a fragment, derivative or variant of such a
polypeptide.
TABLE-US-00001 TABLE ONE Proposed function of SEQ rpl encoded
Position in R. equi ID locus Identifier protein 103S NO rplA
REQ_18350 Prepilin Position 1938280-1939068 1 peptidase
(complement) in 103S genome rplB REQ_18360 Pilin subunit Position
1939395-1939601 2 in 103S genome rplC REQ_18370 Minor pilin
Position 3 protein 1939683.-1940084 in 103S genome rplD REQ_18380
Putative Position 1940093-1941037 4 lipoprotein 1940084 in 103S
genome rplE REQ_18390 Pilus assembly Position 1941047-1941784 5
protein in 103S genome rplF REQ_18400 Pilus assembly Position
1941781-1942980 6 ATPase in 103S genome rplG REQ_18410 Secretion
Position 1942977-1944374 7 apparatus in 103S ATPsae genome rplH
REQ_18420 Secretion Position 1944371-1946239 8 apparatus in 103S
integral genome membrane protein rplI REQ_18430 Secretion Position
1946262-1947152 9 apparatus in 103S integral genome membrane
protein
[0009] In embodiments of the invention, the polypeptide or
derivative or variant or fragment thereof can be encoded by a
polynucleotide sequence comprising a polynucleotide sequence of a
gene as listed in Table 2
TABLE-US-00002 TABLE TWO Proposed function of SEQ rpl encoded
Position in R. equi ID locus Identifier protein 103S NO rplA
REQ_18350 Prepilin Position 1938280-1939068 1 peptidase
(complement) in 103S genome rplB REQ_18360 Pilin subunit Position
1939395-1939601 2 in 103S genome rplC REQ_18370 Minor pilin
Position 3 protein 1939683.-1940084 in 103S genome rplD REQ_18380
Putative Position 1940093-1941037 4 lipoprotein 1940084 in 103S
genome rplE REQ_18390 Pilus assembly Position 1941047-1941784 5
protein in 103S genome rplH REQ_18420 Secretion Position
1944371-1946239 8 apparatus in 103S integral genome membrane
protein rplI REQ_18430 Secretion Position 1946262-1947152 9
apparatus in 103S integral genome membrane protein
[0010] In particular embodiments the polypeptide or a derivative or
variant or fragment thereof can be encoded by a polynucleotide
sequence comprising a polynucleotide sequence of a gene selected
from rplB (SEQ ID NO 2), rplC (SEQ ID NO 3), or rplD (SEQ ID NO 4).
In an alternative embodiment, the polypeptide or a derivative can
be encoded by a polynucleotide sequence comprising a polynucleotide
sequence of a gene selected from rplB (SEQ ID NO 2), rplA (SEQ ID
NO 1) or rplE (SEQ ID NO 5).
[0011] In embodiments of the invention, the polypeptide or a
derivative or fragment thereof is encoded by a polynucleotide
sequence comprising a polynucleotide sequence of a gene selected
from the list of genes of Table 1, more preferably selected from
the list of genes of Table 2.
[0012] In embodiments of the invention, the polypeptide or a
derivative or fragment or variant thereof is encoded by a
polynucleotide sequence consisting essentially of or consisting of
a polynucleotide sequence of a gene selected from the list of genes
of Table 1, more preferably selected from the list of genes of
Table 2.
[0013] In embodiments, the polypeptide is encoded by a
polynucleotide sequence comprising the polynucleotide sequence of a
gene encoding Rpl pilin
ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA
GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC
GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT
CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG
ACGATCCCGGCGGCGAGTAG (SEQ ID NO 2).
[0014] In embodiments, the polypeptide is encoded by a
polynucleotide sequence consisting essentially of or consisting of
the polynucleotide sequence of a gene encoding Rpl pilin
ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA
GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC
GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT
CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG
ACGATCCCGGCGGCGAGTAG (SEQ ID NO 2).
[0015] In embodiments, the polypeptide is encoded by a
polynucleotide sequence comprising a fragment of the polynucleotide
sequence of a gene encoding Rpl pilin
ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA
GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC
GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT
CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG
ACGATCCCGGCGGCGAGTAG (SEQ ID NO 2)
[0016] wherein the polypeptide encoded by the fragment is a
biologically active immunogenic fragment of a polypeptide encoded
by the polynucleotide sequence comprising the polynucleotide
sequence of the gene encoding Rpl pilin
ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA
GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC
GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT
CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG
ACGATCCCGGCGGCGAGTAG (SEQ ID NO 2).
[0017] In embodiments, a derivative or fragment or variant can be
an immunogenic derivative or fragment or variant that can provide
an immune response in which antibodies with binding specificity to
at least one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, and 9 are
generated for example antibodies cross-reactive to the biologically
active immunogenic fragment and at least one of SEQ ID NO 1, 2, 3,
4, 5, 6, 7, 8 and 9.
[0018] In particular embodiments such fragments, derivatives or
variants can functionally provide a pilus in R. equi. Such
derivatives, fragments or variants can be biologically active
derivatives, fragments or variants.
[0019] In embodiments the Rpl pilin polypeptide (RplB) can comprise
an amino acid sequence
MNLFFANLYLMGLDVKDRLTRDDRGATAVEYGLMVAGIAMVIIVAVFAFG
DKITDLFDGFNFDDPGGE (SEQ ID NO 10).
[0020] In embodiments, a polypeptide of the invention can consist
of an amino acid sequence
MNLFFANLYLMGLDVKDRLTRDDRGATAVEYGLMVAGIAMVIIVAVFAFG
DKITDLFDGFNFDDPGGE (SEQ ID NO 10).
[0021] In embodiments a polypeptide of the invention can comprise
DKITDLFDGFNFDDPGGE (SEQ ID NO 11) or can be a variant thereof
wherein such variant has at least 1, at least 2, at least 3, at
least 4, at least 5, at least 6, at least 7, at least 8, at least
9, at least 10 amino acids different to that of SEQ ID NO 11.
Substituted amino acids may suitably be conservative or non
conservative amino acids. Alternatively, the variant may include
insertions or deletions. Suitably, in embodiments a variant can
demonstrate analogous biological function as a RplB pilin subunit
or SEQ ID NO 11. In embodiments, a conserved variant may be
provided by amino acid sequences comprising DKITDLFDGFNFDDPGGE (SEQ
ID NO 11) wherein amino acids as shown are replaced by amino acids
which are structurally conservative. For example, an aliphatic
amino acid (alanine, serine, valine, leucine, isoleucine or the
like) can be substituted with another suitable aliphatic amino
acid, a hydrophobic amino acid (tyrosine, phenylalanine,
tryptophan) can be substituted by another hydrophobic amino acid or
a charged amino acid can be substituted by another charged amino
acid. In such conserved variants, additional amino acids may be
substituted.
[0022] In embodiments a polypeptide of the invention can comprise
the amino acid sequence DKITDLFDGFNFDDPGGE (SEQ ID NO 11). In
embodiments a polypeptide of the invention consists of, or consists
essentially of the amino acid sequence DKITDLFDGFNFDDPGGE (SEQ ID
NO 11).
[0023] A polypeptide of the invention may be provided using
recombinant means or may be a synthetic polypeptide or may be
extracted from R. equi bacteria, R. equi culture supernatant or
from biological material infected with R. equi. In embodiments an
isolated immunogenic polypeptide of the invention is expressed at
the bacterial cell surface of a R. equi, or is secreted from R.
equi.
[0024] In embodiments, a polypeptide of the invention, or a
fragment, derivative or variant thereof comprises an amino acid
sequence of at least one polypeptide selected from the group
consisting of the list provided by Table 3 or as set out in the
sequences of FIG. 9.
TABLE-US-00003 TABLE THREE Rpl protein Identifier Proposed function
SEQ ID NO RplA REQ_18350 Prepilin peptidase 12 product RplB
REQ_18360 Pilin subunit 13 product RplC REQ_18370 Minor pilin
protein 14 product RplD REQ_18380 Putative lipoprotein 15 product
RplE REQ_18390 Pilus assembly 16 product protein RplF REQ_18400
Pilus assembly 17 product ATPase RplG REQ_18410 Secretion apparatus
18 product ATPsae RplH REQ_18420 Secretion apparatus 19 product
integral membrane protein RplI REQ_18430 Secretion apparatus 20
product integral membrane protein
[0025] All of the polypeptides shown in Table 3 are encoded in the
rpl locus and are part of the R. equi Rpl pilus biogenesis
machinery.
[0026] In embodiments a polypeptide of the invention can be encoded
by an R. equi. strain isolated from horses. In embodiments the
polypeptide can be isolated from horses and can be from a virulent
strain of R. equi. In embodiments, polypeptides of the invention
can be made synthetically or recombinantly using techniques which
are widely available in the art.
[0027] The polypeptide of the invention may be optionally linked to
an immunogenic carrier. Said immunogenic carrier may be a
heterologous polypeptide, lipid, liposome, or another acceptable
carrier molecule. Suitably, a polypeptide of the invention may be
linked to the immunogenic carrier by chemical coupling or a
polypeptide of the invention may be expressed as a fusion protein
with the immunogenic carrier. A polypeptide of the invention,
and/or a biologically active and/or immunogenic fragment, or
derivative, or variant thereof, can be provided in an immunogenic
composition, for example to raise antisera or monoclonal antibodies
for passive immunisation, or as a vaccine. Alternatively, a
polypeptide of the invention, fragment, derivative or variant
thereof may be useful in an assay to detect antibodies specific for
the polypeptide, including diagnostic assays. As set out herein, in
embodiments, a derivative of a polypeptide of the invention can be
a composite of specific polypeptide sequences of the invention, for
example composites of SEQ ID NO 10, SEQ ID NO 11 and a polypeptide
as set out in Table 3 or fragments thereof, or nucleotide sequences
for example as set out at Table 1 or Table 2 disclosed herein. In
embodiments, the nucleic acid sequences can be used to form
concatemers and may be used to provide polypeptide sequences, for
example relevant epitopes may be put in tandem or provided in
multiples of 3, 4, 5, 6, or greater than 10, greater than 20 or
more. Further, in embodiments a derivative can include a scrambled
or chimeric polypeptide containing combinations of different
relevant Rpl polypeptides. In such embodiments the combinations of
relevant Rpl polypeptides can be provided in multiples of 2, 3, 4,
5, 6, or greater than 10, greater than 20 or more.
[0028] It is important to note that even with knowledge of the
genome of R. equi strain 103S, it would not be apparent that R.
equi produced pili appendages or that the nine-gene locus
encompassing nucleotide positions 1,938,280 to 1,947,152 (locus
tags REQ18350-430) encoded a pilus biogenesis apparatus responsible
for the production of R. equi pili involved in virulence and host
colonisation. Pili are widespread among bacteria and can serve many
functions unrelated to virulence. For example pili can facilitate
attachment of bacteria to environmental surfaces such as soil
particles, biofilm formation, be mediators of bacterial motility or
enable adhesion to other bacteria. As will be appreciated,
depending on pili function, in some instances, pili may not provide
an immunogenic determinant suitable for vaccine development or be
able to act as a diagnostic marker.
[0029] Using visualisation by electron microscopy and genetic
molecular analysis, the inventors demonstrated for the first that
R. equi produces pili appendages or fimibriae, identified that the
rpl locus R. equi encodes the pilus biogenesis apparatus, and
further determined that proteins of R. equi pili are major
virulence factors involved in host colonisation and that they are
major immunodominant antigens. The latter determination would not
have been suggested from sequence data alone.
[0030] According to a second aspect of the present invention there
is provided an isolated or recombinant nucleic acid encoding a
polypeptide associated with pilus formation in R. equi. In
embodiments of the invention there is provided an isolated or
recombinant nucleic acid comprising a polynucleotide sequence
comprising or consisting of a sequence as set forth in any one of
SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5,
SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, and SEQ ID NO 9 or a variant
or derivative or fragment thereof, for example as illustrated in
the sequences of FIG. 10.
[0031] Due to the known degeneracy of the genetic code, a
polynucleotide sequence which differs from those indicated by any
one of SEQ ID 1, 2, 3, 4, 5, 6, 7, 8 or 9 can encode an active
immunogenic derivative, variant or fragment and/or a biologically
active derivative, variant or fragment of a polypeptide of the
invention. In embodiments, a polynucleotide sequence which encodes
such a derivative, fragment or variant sequence or an immunogenic
biologically active derivative or fragment can result from silent
mutations (e.g., occurring during PCR amplification), or nucleotide
substitutions, deletions or insertions or the like or can be the
product of deliberate mutagenesis of a native sequence. Variant
polypeptides may be encoded by variant polynucleotide sequences
having sequence homology (identity) of greater than at least 85%,
86%, 87%, 88%, 89%, preferably at least 90%, 91%, 92%, 93%, 94%,
and more preferably 95%, 96%, 97%, 98%, 99% but less than 100%
contiguous nucleotide sequence homology to any one of SEQ ID NO 1,
2, 3, 4, 5, 6, 7, 8, or 9 or fragments thereof. A variant
polypeptide may be encoded by a polynucleotide sequence including
nucleotide bases not present in the corresponding wild type nucleic
acid molecule and/or internal deletions relative to the
corresponding wild type nucleic acid molecule, such as SEQ ID NOs
1, 2, 3, 4, 5, 6, 7, or 8. Polynucleotide sequences encoding
fragments of a polypeptide of the invention may be greater than 30
nucleotides in length, greater than 50 nucleotides in length,
greater than 100 nucleotides in length, or greater than 150
nucleotides in length. The invention also provides isolated nucleic
acids useful in the production of polypeptides. Suitably said
biologically active immunogenic derivative, fragment or variant can
elicit an immune response wherein the antibodies generated to said
derivative, fragment or variant have a binding specificity to any
one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8 or 9. In embodiments, there
can be provided a polynucleotide sequence comprising or consisting
of a sequence as set out in any one of SEQ ID NO 1, SEQ ID NO 2,
SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7,
SEQ ID NO 8, and SEQ ID NO 9.
[0032] In further embodiments there is provided an isolated or
recombinant nucleic acid comprising a polynucleotide sequence
comprising or consisting of a sequence as set forth in any one of
SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5,
SEQ ID NO 8, and SEQ ID NO 9. In additional embodiments, there is
provided an isolated or recombinant nucleic acid comprising a
polynucleotide sequence comprising or consisting of a sequence as
set forth in any one of SEQ ID NO 2, SEQ ID NO 3, and SEQ ID NO 4.
In specific embodiments there is provided an isolated or
recombinant nucleic acid comprising a polynucleotide sequence
comprising or consisting of a sequence as set forth in SEQ ID NO
2.
[0033] Polypeptides of the invention or a biologically active
immunogenic fragment, derivative, or variant thereof may be
prepared as a pharmaceutical preparation or composition. Such
preparations will comprise the polypeptide or a biologically active
immunogenic fragment, derivative, or variant thereof and a suitable
carrier, diluent or excipient. These preparations may be
administered by a variety of routes, for example, oral, buccal,
topical, intramuscular, intravenous, subcutaneous, intranasal or
the like.
[0034] In a third aspect of the present invention, there is
provided a composition comprising a polypeptide or antibody
according to the invention, or a biologically active immunogenic
fragment, derivative, or variant thereof, together with a
pharmaceutically acceptable carrier. A carrier and/or excipient
useful in a composition of the present invention will generally not
inhibit to any significant degree a relevant biological activity of
the polypeptide or antibody of the invention. Alternatively, or in
addition, the carrier or excipient can comprise a compound that
enhances uptake and/or delivery and/or efficacy of the polypeptide
and/or antibody as described herein. Alternatively, or in addition,
the carrier or excipient can comprise a compound that enhances the
activity of a polypeptide and/or antibody as described herein
and/or reduces inhibition of said polypeptide or antibody by
degradative enzymes in the site of administration and/or on route
to the site of action and/or at the site of action. For example,
the carrier or excipient may comprise a protease inhibitor and/or a
DNase inhibitor and/or an RNase inhibitor to thereby enhance the
stability of a polypeptide and/or antibody as described herein
above or nucleic acid encoding same.
[0035] As will be apparent to the person skilled in the art based
on the foregoing description, the methods of the present invention
further comprise providing, producing or obtaining a composition
comprising a polypeptide and/or an antibody or nucleic acid
encoding said polypeptide. Suitable methods for producing such
compositions will be apparent to the skilled artisan based on the
disclosure herein. A polypeptide can also be delivered with other
relevant antigens in a polyvalent protein vaccine.
[0036] In certain further aspects, the present invention provides
an antibody which has binding specificity to at least one of the
polypeptides of the invention or a fragment, derivative, or variant
thereof, or an antigen binding fragment of said antibody.
Accordingly, in a fourth aspect of the invention there is provided
an antibody which specifically binds to a polypeptide of the
invention or an epitope, fragment, derivative or variant thereof.
Antibodies of the present invention may confer protection against
infection with R. equi. Additionally or alternatively, an antibody
can specifically bind to a polypeptide of the invention or can bind
to an epitope of the pili provided on R. equi or an R. equi antigen
of the pili and whilst not conveying protection against infection
with R. equi, may be a useful in an immunoassay for the detection
of polypeptides of the invention or for diagnosis of R. equi
infection.
[0037] In certain embodiments, the antibody can be a polyclonal
antibody. Alternatively, the antibody can be a monoclonal antibody,
a chimeric antibody, or a synthesized or a synthetic antibody.
Methods for producing a polyclonal and monoclonal antibodies are
well known in the art and an antibody provided against a
polypeptide of the pili is described herein.
[0038] In certain further aspects, the present invention further
extends to a method of producing an antibody which specifically
binds to at least one polypeptide of the present invention, or a
biologically active and/or immunogenic fragment, derivative or
variant thereof, said method comprising: [0039] (i) immunising a
host with a polypeptide or a fragment, derivative, or variant
thereof as described herein according to any embodiment, and [0040]
(ii) recovering antibodies generated by the host against said
polypeptide or a fragment, derivative, or variant thereof.
[0041] The present invention also provides a method for producing
an antibody that binds to an antibody which specifically binds to
at least one polypeptide of the present invention or a fragment,
derivative, or variant thereof (i.e., a method for producing an
anti-idiotypic antibody), said method comprising: [0042] (i)
immunising a host with an antibody that binds to a polypeptide of
the invention or a fragment, derivative, or variant thereof or an
antigen binding fragment of said antibody, [0043] (ii) identifying
antibodies generated by the host against an antigen binding site of
said antibody; and [0044] (iii) recovering the antibodies
identified at (ii).
[0045] The present invention also provides an anti-idiotypic
antibody that selectively binds to an antibody that binds to a
polypeptide or a fragment, derivative, or variant thereof as
described herein or an antigen binding fragment of said
antibody.
[0046] In a fifth aspect of the present invention there is provided
a composition comprising an antibody of the invention together with
a pharmaceutical carrier.
[0047] The invention also provides vectors comprising nucleic acids
of the invention and cells comprising such vectors.
[0048] In the sixth aspect of the invention there is provided a
construct comprising a nucleic acid molecule which encodes a
polypeptide of the invention, for example an isolated nucleic acid,
or a fragment, derivative, or variant thereof operably linked to a
promoter which is functional to allow transcription of the nucleic
acid sequence and the expression of an R. equi polypeptide of the
invention.
[0049] The present invention also provides a process for producing
a polypeptide or a fragment, derivative, or variant thereof as
described herein according to any embodiment, said method
comprising culturing a cell comprising a nucleic acid encoding a
polypeptide or a fragment, derivative, or variant thereof of the
present invention operably linked to a promoter under conditions
suitable for expression of the polypeptide or a fragment,
derivative, or variant thereof. A suitable nucleic acid may
comprise a polynucleotide sequence or fragment thereof of a gene
selected from Table 1, or more preferably Table 2. In one example,
the method additionally comprises recovering the polypeptide from
the cell culture, e.g., from the medium in which the cell is
cultured.
[0050] In embodiments the present invention provides a method of
producing a polypeptide or a fragment, derivative, or variant
thereof of the invention, said method comprising the steps of:
[0051] (i) culturing a host cell comprising a nucleic acid encoding
a polypeptide of the present invention or a vector encoding the
same, and [0052] (ii) recovering the polypeptide of the present
invention from the host cell or culture medium.
[0053] In embodiments, the construct comprises an isolated nucleic
acid which encodes a polypeptide of the invention or a fragment,
derivative, or variant thereof operably linked to a promoter which
is functional in a host cell to allow transcription of the nucleic
acid sequence and the expression of a R. equi polypeptide of the
invention.
[0054] In alternative embodiments, the construct comprises an
isolated nucleic acid which encodes a polypeptide of the invention
or a fragment, derivative, or variant thereof operably linked to a
promoter which is functional in a heterologous host system, for
example an attenuated vaccinal strain, including, but not limited
to, a microbial system, a virus, a parasite, an attenuated pathogen
or normal or immuno-stimulating microbiota. Suitably, the
heterologous host system construct may be delivered as a live
vaccine alone or in combination with other relevant protective
antigens in a polyvalent vaccine.
[0055] In embodiments, the construct can comprise a nucleic acid
comprising a polynucleotide sequence of a gene selected from at
least one gene identified by Table 1, more preferably a gene
selected from Table 2, operably linked to a promoter.
[0056] In embodiments, the construct can comprise a nucleic acid
sequence comprising a polynucleotide sequence of SEQ ID NO 1, 2, 3,
4, 5, 6, 7, 8 or 9, more preferably a polynucleotide sequence which
can encode SEQ ID NO 10 or 11.
[0057] In a seventh aspect of the invention there is provided a
construct of the sixth aspect of the present invention in
combination with a pharmaceutical carrier.
[0058] In an eighth aspect of the present invention there is
provided a composition capable of treating or preventing a disease
caused by R. equi, comprising one or more surface-associated (a
polypeptide naturally associated to the surface structures or on
the outer surface of R. equi.) or secreted polypeptides of R. equi
wherein said polypeptides form pili of R. equi. In embodiments the
composition can be a vaccine capable of preventing a disease caused
by R. equi, which results in the production of antibodies against a
polypeptide of R. equi which can form the pili of R. equi and
wherein the polypeptide is reactive against antibodies or immune
cells recovered from animals repeatedly infected with R. equi.
[0059] In embodiments, the polypeptide of R. equi which can form
the pili of R. equi, wherein the polypeptide is reactive against
antibodies and/or immune cells recovered from animals repeatedly
infected with R. equi comprises the amino acid sequence encoded by
a polynucleotide sequence of a gene selected from Table 1, or more
preferably Table 2 or is an immunogenic fragment or variant or
derivative of such a polypeptide.
[0060] In embodiments of the invention, the subject for which the
vaccine can be administered is a foal and immunisation results in
an immune response which inhibits or prevents R. equi infection and
results in the production of antibodies employed as an
immunogen.
[0061] In embodiments the subject to which the vaccine is
administered can be a horse and immunisation results in an immune
response which inhibits or prevents R. equi., or in the production
of antibodies to the polypeptide employed as an immunogen.
[0062] While the invention is particularly directed to polypeptide
suitable as antigen in a vaccine for use in horses or foals, it
will be clearly understood that it is applicable to any other
animal which is susceptible to infection with R. equi, including
humans, pigs, cattle, sheep, goats, lama, cats or animals which
have a similar biology and would be understood to share a high
degree of genomic similarity to horses. It will also be appreciated
that the diagnostic, therapeutic and prophylactic aspects of the
invention are also applicable to subjects which have been exposed
to an animal infected with R. equi, or an environmental source
contaminated with R. equi such as faeces, soil, or the like.
[0063] According to a ninth aspect of the present invention there
is provided a method of treating or preventing a disease or
condition caused by R. equi comprising the step of administering a
polypeptide of the invention or a fragment, derivative, or variant,
an antibody, a nucleic acid, composition and/or a vaccine of the
invention to subjects suffering from, or suspected to be suffering
from, or at risk of a condition mediated by R. equi.
[0064] There is provided the use of a polypeptide of the invention
or a biologically active and/or immunogenic fragment, derivative,
or variant, an antibody, a nucleic acid, composition and/or a
vaccine of the invention in the preparation of a medicament for the
treatment of a condition mediated by R. equi. In embodiments the
treatment may be prophylactic treatment to prevent or inhibit
infection.
[0065] There is provided a polypeptide of the invention or a
fragment, derivative, or variant, an antibody, a nucleic acid,
composition and/or a vaccine of the invention for use in the
treatment of a condition mediated by R. equi. In embodiments the
treatment may be prophylactic treatment to prevent or inhibit
infection.
[0066] According to a tenth aspect of the present invention there
is provided a method of detecting R. equi comprising the step of
detecting a polypeptide of the invention or a fragment, derivative,
or variant, or an antibody of the invention in a sample, or a
polynucleotide of the invention which can encode a polypeptide of
the invention or fragment thereof. In embodiments, a sample may be
a soil sample.
[0067] In embodiments, there is provided a method of diagnosing a
disease or condition caused by R. equi comprising the step of
detecting a polypeptide of the invention or a fragment, derivative,
or variant, or an antibody of the invention in a biological sample
from a subject suffering from, suspected to be suffering from, or
at risk of such a condition, or a polynucleotide of the invention
which can encode a polypeptide of the invention or fragment
thereof.
[0068] Detection of a polypeptide or an antibody of the invention
may be achieved by a variety of methods, including but not limited
immunoassay methods such as radioimmuno assay, enzyme linked
immunoabsorbent assays (ELISA), chemiluminescence assays,
immunohistochemistry, immunoblotting, for example Western blotting,
immunofluorescence and mass spectrometry. An example of use of an
antibody to detect a polypeptide of a R. equi pili (RplB) is
provided in the Examples herein.
[0069] Suitably, detection of antibodies with binding specificity
to a polypeptide encoded by any one of SEQ ID NO 1, 2, 3, 4, 5, 6,
7, 8, or 9 may be used as a test for R. equi in horses. In
embodiments, PCR testing for nucleic acids encoding a polypeptide
of the pili, for example as encoded by any one of SEQ ID NO 1, 2,
3, 4, 5, 6, 7, 8, or 9 may be used as a test for R. equi,
particularly where a quantitative detection is preferred. Based on
the nucleic acid sequence data provided herein, suitable primers or
probes for use in the detection of nucleic acid sequences which can
encode polypeptides of the pili of R. equi could be provided as
would be understood in the art. As will be understood, suitably, in
embodiments, said probes or primers can hybridise to the nucleic
acid sequences encoding peptides associated with pilus formation,
preferably any one of SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8, or 9, under
stringent conditions. Hybridisation refers to the binding,
duplexing, or hybridizing of a molecule only to a particular
nucleotide sequence under stringent conditions when that sequence
is present in a complex mixture (e.g., total cellular) DNA or RNA.
Stringent hybridisation occurs when a nucleic acid binds the target
nucleic acid with minimal background. Typically, to achieve
stringent hybridisation, temperatures of around 1.degree. C. to
about 20.degree. C., more preferably 5.degree. C. to about
20.degree. C. below the Tm (melting temperature at which half the
molecules dissociate from their partner) are used. However, it is
further defined by ionic strength and pH of the solution. An
example of highly stringent wash conditions is 0.15 M NaCl at
72.degree. C. for about 15 minutes. An example of a stringent wash
condition is a 0.2.times.SSC wash at 65.degree. C. for 15 minutes
(see, Sambrook and Russell, infra, for a description of SSC
buffer). Often, a high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example of a
medium stringency wash for a duplex of, for example, more than 100
nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An
example of a low stringency wash for a duplex of for example more
than 100 nucleotides, is 4-6.times.SSC at 40.degree. C. for 15
minutes. For short probes (for example about 10 to 50 nucleotides),
stringent conditions typically involve salt concentrations of less
than about 1.5 M, more preferably about 0.01 to 1.0 M, Na ion
concentration (or other salts) at pH 7.0 to 8.3, and the
temperature is typically at least about 30.degree. C. and at least
about 60.degree. C. for long probes (for example, >50
nucleotides). Detection of a polynucleotide of the invention may be
by any suitable means, for example using PCR, a microarray or the
like as would be known in the art.
[0070] In an eleventh aspect of the present invention there is
provide a kit to detect R. equi wherein the kit comprises a
polypeptide or antibody of the invention or a nucleic acid probe.
In embodiments a kit can comprise a polypeptide or antibody of the
invention.
[0071] In embodiments, the kit is for use in the method of
diagnosing a disease or condition caused by R. equi wherein the kit
comprises a polypeptide or antibody of the invention or a nucleic
acid probe. In embodiments a kit can comprise a polypeptide or
antibody of the invention.
[0072] In embodiments, the kit can include a solid support, for
example a test strip, plastic bead or the like to which polypeptide
or antibody of the invention can be coated. The kit may include a
detection antibody capable of binding to a polypeptide or antibody
of the invention which comprises a detectable label or binding site
for a detectable label. Suitably a labelling molecule can include
an enzyme, fluorescent label or radiolabel. Binding sites for
detectable labels include avidin, biotin, streptavidin and the
like.
[0073] Additionally, the kit can include instructions for using the
kit to practise the present invention. The instructions should be
in writing in a tangible form or stored in an electronically
retrievable form. A further aspect of the present invention
provides a method of screening immunogenic R. equi polypeptides of
the invention or a fragment, derivative, or variant thereof to
determine if a test agent can bind to said polypeptide comprising
the steps: providing a candidate immunogenic R. equi polypeptide of
the invention or a fragment, derivative, or variant thereof,
providing a test agent to the candidate immunogenic R. equi
polypeptide and determining whether said test agent can bind to
said candidate immunogenic R. equi polypeptide.
[0074] A test agent which can bind to a R. equi polypeptide of the
invention may inhibit the activity of said polypeptide, minimise
its secretion or inhibit its ability to form functional pili. In
embodiments, such a test agent may be a useful therapeutic.
[0075] The present invention also provides the use of a polypeptide
or a fragment, derivative, or variant thereof or an antibody as
described herein in medicine.
[0076] In a twelfth aspect, the present invention provides the use
of a polypeptide of the invention or a fragment, derivative, or
variant thereof, an antibody, composition and/or vaccine of the
invention in the treatment or prevention of a disease or condition
caused by R. equi.
[0077] In one embodiment of the invention, a method of treatment
comprises the steps:
[0078] (i) identifying a subject suffering from a disorder
associated with or R. equi or at risk of developing R. equi;
[0079] (ii) administering a polypeptide, or composition as
described herein to said subject.
[0080] In another embodiment, the invention provides a method of
treatment comprising administering or recommending a polypeptide,
or a fragment, derivative, or variant thereof or an antibody or
composition as described herein to a subject previously identified
as having R. equi infection or suffering from a condition
associated with R. equi infection. The invention may also provide a
method of treatment of a subject in need thereof, said method
comprising: [0081] (i) identifying a subject suffering from a
disorder associated with or R. equi or at risk of developing R.
equi; [0082] (ii) obtaining a polypeptide or a fragment,
derivative, or variant thereof as described herein according to any
embodiment, [0083] (iii) formulating the polypeptide or antibody
with a suitable carrier and/or excipient to form a composition
wherein said composition is in an amount sufficient to reduce or
prevent or inhibit R. equi infection or suffering from a condition
associated with R. equi infection and [0084] (iv) administering
said composition to said subject.
[0085] In a further embodiment there is provided a method of
treatment of a subject in need thereof, said method comprising:
[0086] (i) identifying a subject suffering from a disorder
associated with or R. equi. or at risk of developing R. equi.;
[0087] (ii) obtaining a polypeptide or a fragment, derivative, or
variant thereof or an antibody as described herein according to any
embodiment, [0088] (iii) formulating the polypeptide or antibody
with a suitable carrier and/or excipient to form a composition
wherein said composition is in an amount sufficient to reduce or
prevent or inhibit R. equi infection or suffering from a condition
associated with R. equi infection and [0089] (iv) recommending
administration of a composition at (iii).
[0090] In embodiments a polypeptide on the invention can be
provided to a subject to generate a protective immune response in
the subject. In particular embodiments the polypeptide may act as a
vaccine.
[0091] Sequences identified in the patent application include:
TABLE-US-00004 SEQ ID NO 1 rpIA REQ_18350> - 3104103:3104891
GTGATCGTCGCAGCGGGCGTCGGCGCCGCACTCCTGGGTATCCTCG
CCGGGGCGTTCGCGAACAGTGCGATCGACCGCGTGCGCCTGGAGA
CCGCGTGCGCCGAGCCGAAGTCGACCCCCACCGGCTCAACCCCGC
CGCCCCCCTCCCCTGCGTCCGCGGTAGCCACCCGGATCGCGATGAT
CGACACCATCACGCGACGACACGACATCAGTGCCCGCCGCGTGCTC
GTCGAACTCGCAACGGCCCTCCTGTTCGTCGGGATCACTCTCCGTCT
CGCCGCTCTCGGTCTTCTCCCGGCAACACCGGCCTATCTCTTGCAAA
CGGCTGCCGAACTTCCTCGTCGTACCGTCGTACCCGATCGTATTCGC
CTGCCTTTCAGTGGGTTCCGTCGTGCCGTTCTGTTCGGGGTCTACTT
CGTACTAGCCCTGATCTATCCGGCCGGCATGGGGTTCGGCGACGTC
AAACTTGCCGGCGTCATCGGCGCCGTCCTCGCCTACCTGTCGTACG
GCACATTGCTCGTCGGGGCGTTTCTCGCGTTCCTGGTGGCCGCACT
CGTCGGCCTGATCATCCTGGTCACCCGTCGCGGTCGGATCGGGACC
ACGATTCCCTTCGGGCCGTACATGATTGCGGCGGCCATCGTTGCGAT
CCTGGCGGCCGATCCGCTGGCGCGCGCGTATCTGGACTGGGCCGC CGCGGCCTGA SEQ ID NO
2 rpIB REQ_18360> - 1939395:1939601
ATGAACCTCTTCTTCGCGAACCTGTACCTCATGGGCTTAGACGTCAA
GGACCGTCTGACCCGTGACGACCGCGGCGCCACTGCGGTCGAGTAC
GGACTGATGGTCGCCGGCATCGCGATGGTGATCATTGTTGCGGTTTT
CGCCTTCGGCGATAAGATTACCGACCTCTTCGATGGCTTCAACTTCG
ACGATCCCGGCGGCGAGTAG SEQ ID NO 3 rpIC REQ_18370> -
1939683:1940084 ATGAAGCGCCTCACTTCCGATTCAGGGGTCGCCGCAGTCGAATTCGC
TCTCGTCGTTCCGATCCTGATCACACTGGTCCTCGGCATCGTGGAGT
TCGGTCGGGGTTACAACGTCCAGAACGCGGTCAGCGCTGCTGCCCG
CGAGGGTGCACGGACGATGGCGATCAAGAAGGATCCGGCGGCGGC
GCGTGCTGCCGTGAAGGGCGCGGGTGTGTTCAGTCCGGCGATCACC
GATGCGGAGATCTGCATCAGCACTTCGGGAACGCAGGGCTGTTCGG
CAACGTCGTGTCCGAGCGGAAGTACCGTGACGCTCACGGTCAGCTA
TCCACTCGAGTACATGACGGGACTCTTTCCCGGTAAGCCGACGCTCA
CCGGCACGGGGGTCATGCGATGCGGTGGGTGA SEQ ID NO 4 rpID REQ_18380> -
1940093:1941037 ATGTCGAATGACGAGCGCGGGGTCGTCGCCGTGCTCGTTGCGATCC
TCATGGTCGTGCTCCTGGGATGTGCTGCGATCTCGGTCGACATCGGT
GCGAACTATGTCGTCAAACGTCAGTTGCAGAACGGGGCCGATGCGG
CTGCGCTCGCCGTAGCTCAGGAATCCAGTTGCAAGGCAGGATCTTCC
GCCTCATCCGTGTCGAGCCTTGTCCAGGCGAACGTCAACAGCTCGTC
GGCTGCAAGTGCGGCGGTGATCGACGGTGTGAAGCGGAAGGTGAC
GGTCACTGCGTCGGCGGTGGGTGACGACGGCCTCGCCGGCCGGAG
GAACGTGTTCGCTCCGGTCCTCGGAGTCGACCGCAGCGAGATCTCG
GCGTCTGCGACTGCAAGCTGCGTGTTTCCCCTCGGGGGGACCGCGG
AACTCCCGCTCACGTTCCACAAGTGCCATTTCGACGAATCCCGCAGT
CTGGACGTGAAGATCCTCGTCGCCTACAACGTGACGGCGCCGCGCT
GCAATGGAACCTCGGGAAATGCGGCACCGGGCAATTTCGGCTGGCT
GCAGGGGGCGAACGGTCGATGCCCGGCGAAGATCGACGCCGCCGT
CTACGCAACACCGGGCGACACCGGTAACAACATTCCGGGGCCGTGC
AAGGACACCATCAAGCAGTTTCAGAATGCCGTCGTGCGGGTCCCGAT
CTACGACGTCGCAGGTGGAACCGGAAGCGGTGGATGGTTTCACGTC
GTCGGTTTGGCTGCCTTCAAGATTCAGGGCTACCGGCTGAGCGGCA
ACCCGGAGTTCAACTGGAACAACGATGTTCACGGGGCGCTGAGTTG
CACCGGCAGCTGTCGCGGCATCATCGGCACCTTCGTGAAGATTGTCA
GCCTCGATTCGGATCTGACGCCGGGAGGGATCGATTTCGGCGTGAG
TACGATCAGCTTGCTCGATTAG SEQ ID NO 5 rpIE REQ_18390> -
1941047:1941784 TTGAGAACCCGAATCATTGCTGCGATCTGTGCGATCGTTCTCGCGGT
CGCGGGAACCCTCGCCCTGATCTCGTATGTACGCGGGGCCGATGCC
CGCGCCCTGGCGGGTACACGCACCGTCGATGTGCTCGTCGCCGATC
AGACGATTCCGAAGAACACTCCCGCTGATTCGCTCGTGGGAATGGTT
GTGGTCAAGAAACTTCCGGAAATGGCGGTGCTACCCGATCGGGTGA
CCAGTCTCGACCAACTGTCCGGCAAGGTCGCGCTGACCGACCTCCT
GCCTGGCGAACAACTGGTCTCGGCGCGATTCGTCGACCCGGCGACC
GCCCGAAGTCAGGACCAGGGAGGAATCCCCGAGGGGATGCAGGAG
GTGACGGTTCTTCTCGAGCCGCAACGCGCACTGGGAGGCCACATCG
CGTCGGGCGATACCGTCGGCGTCTTCATGTCCTTCTCGCCGCCCGT
CAAGAACTACGAAACACATCTGAGATTGCAGAAAGTGCGAGTCACGC
GGGTCCAGGGAACGTTCTCCAACGCCGACGAAGGGGATTCGGCCAC
GGTCGACTCGTCGCCGAGCCCTGCTCCCACCGAGGCCTTTCTCGTC
TCGCTGGCGGTCGACGTGCCGATGGCGGAGCGCGTCGTTTTCGCCG
CGGAGCACGGGACCATCTGGCTTTCCAATGAGCCGCCGAGTTCGAA
CGAGGCCGGGGCATCCGTGGTCTCCCCGGAAGGAGTGTTCCGATGA SEQ ID NO 6 rpIF
REQ_18400> - 1941781:1942980
ATGAGCCGCATCGTCCTGCTGACCGATCGCGACGATTTCGCCCGCC
GCGTGTACCACGCCGCGGACGGCAACCTTCTGGTGTTGCCGGCGCA
GCCGGTTCCCCGGGGGCCGGCGCAGTTGGTCGGGCTCGGCGTGAC
CGTGCAACCAGAAGTTCTCGTTCTCGGTCCGGACGTGCCGGAAGTG
GAGGGCCTCTCCCTCGCCGGCCGGATCGATCATTCGACGCCCGGCA
CCACGGTGGTTCTGGCCAGTGATGCGGGCACCGACGTGTGGTTGCG
GGCGATGCGCGCCGGCGTGCGGGACGTGATGTCGCCGGAGGCGGA
GATCGCGGACGTTCGTGCGGTACTCGATCGAGCGGGCCAGGCCGCA
CTGGCGCGACGTCAGGGGGCGAGTGCACCGGCGGAGCAGCATGCG
GTTCAAGGGAAGGTCATCGTGGTCGCGTCGCCGAAAGGCGGAACCG
GAAAGACCACCGTTGCGACGAATCTTGCAGTAGGACTCGCGGCGGC
AGCGCCTCACTCGACGGTGTTGGTGGACCTCGACGTGCAGTTCGGG
GACGTTGCCAGTGCTCTCCAGTTGGTTCCGGAACATTGCCTGACCGA
CGCCGTCGCGGGCCCGGCCAGCCAGGACATGATCGTCCTCAAGACC
GTCCTGACACCCCATTCCACAGGACTGCATGCGCTGTGTGGGTCGG
ACTCGCCCGCGGCGGGCGACAGCATCACCGGCGAGCAGGTGAGCA
CTCTGCTGACGCAGTTGGCGGCCGAATTCCGGTACGTGGTCGTCGA
CACCGCGCCCGGTTTGCTCGAACACACCCTGGCGGCGCTCGACCTT
GCTACCGACGTCGTGTTGGTGTCGGGTATGGACGTGCCCAGCGTCC
GCGGGATGCACAAGGAACTGCAATTGCTGACGGAGCTGAATCTGGG
TCCGGTCGTGCGGCATGTCGTGCTCAACTTTGCGGATCGACGCGAG
GGGCTGACGGTCCAGGACATCCAGAACACCATCGGGGTCCCCGCCG
ATATCGTGATCAAGCGCTCGAAAGCCGTTGCCCTCTCGACGAACCGG
GGGGTTCCACTGCTTCAGAACCCGGGTCGGGATCGCACTGCGAAAG
AGTTGTGGCGACTCGTCGGCCGTATCGATCCGGCTCCCGATACCGC
CAAGGGTGGACGCGCGCGGCATCGGGCAGCCGAGGCGGTGGGTGC GAAATGA SEQ ID NO 7
rpIG REQ_18410> - 1942977:1944374
ATGAGACTGTCCCAACGGCTCGAGGCCGTGCGCGGAGCCGCACCC
GTCGAAGCCGCCGCACCGATCCCGCCGGGGAAGCAGGGGAAGGCG
AAAACGTCCCTCCCTCCGGCCGACGCTCTCGCCGAACTGAAGGACC
GTGCGAGTGCGGCCCTGTACACCCGGATCGGCACCCGCTTCAACGA
CTCCTCGTTGAGCGAGGAGCAACTGCATCTCCTGGTCCGTGAGGAA
CTGGCCGAAATCGTGGAGCAGGAGACGACGCCACTCACCTTCGACG
AACGGCAGCGCCTGCTCCGTGAGGTTGCCGACGAGGTACTGGGGCA
CGGACCGCTCCAGCGGCTACTGGAGGACCCGTCGGTCACCGAGATC
ATGGTCAACAGCCACGACATGGTCTACGTCGAGCGGGACGGCACCC
TCGTCCGCAGCTCCGCGCGATTCGCGGACGAGGCGCACCTGCGTCG
CGTGATCGAACGCATCGTTTCCGCCGTCGGTCGACGGATCGACGAA
TCGTCCCCGCTCGTGGATGCACGCTTGGCGGATGGCTCCCGTGTCA
ACGCGGTGATCCCACCGCTCGCATTCAACGGCTCCTCGCTCACCATT
CGAAAGTTCTCGAAAGATCCGTTCCAGGTCGACGATCTCATCGCCTT
CGGCACTCTCTCGCACGAGATGGCCGAACTGCTCGACGCGTGTGTG
CAGGCGCGACTGAACGTCATCGTCTCGGGCGGCACGGGCACGGGG
AAGACGACGCTGCTCAACGTGCTCTCGTCGTTCATTCCGGAAGGGGA
GCGGATCGTCACCATCGAGGACGCCGTGGAACTGCAACTTCAGCAG
GACCACGTCGTACGGTTGGAGAGCCGACCGCCGAACATCGAGGGCA
AGGGTGCCGTCACCATCCGCGACCTGGTGCGGAACTCGCTGCGTAT
GCGTCCCGACCGCATCGTGGTGGGGGAGTGTCGCGGAGGCGAGAG
TCTCGACATGCTGCAAGCGATGAACACCGGTCACGACGGGTCGCTG
TCGACGGTGCATGCGAATTCGCCCCGTGACGCCATCGCGCGCTTGG
AGACGCTCGTGTTGATGGCCGGCATGGACCTGCCGTTGCGGGCGAT
CCGGGAGCAGATTGCTTCGGCGGTCGACGTGATCGTGCAGCTCACT
CGACTACGTGACGGCACTCGGCGAGTGACCCACGTGACCGAGGTCC
AGGGCATGGAGGGTGAGATCGTCACCCTGCAGGATGCCTTCCTGTT
CGACTACAGCGCCGGCGTCGACGCGCGCGGGCGATTCCTCGGCAG
ACCGCAGCCGACCGGAGTGCGGCCGCGGTTCACCGACAGATTCCGA
GATCTCGGTATTGCTTTGTCGCCGAGTGTTTTCGGGGTGGGAGAACC CTCCCGGGGGCGGGTATGA
SEQ ID NO 8 rpIH REQ_18420> - 1944371:1946239
ATGAGCCGGTGCGTGGTGGCCGTCGTGCTCGCCCTCGGTGCGGGT
GTTCTGGGAATTCCCGCCGTAGCCGCGGCGGCCGAGGAGGCTGTCC
AGGTCTCGGCGGTCGACACGACCCGGTTTCCCGACATCGAGGTGTC
CATCCTCGCGCCGCCCGGTATCGAAGGGCAGGCGATCGATCCGGGA
ACGTTCGCGCTCACCGAGGGCGGCGTGCCGCGAGAGATCGAGGTC
AGGCAGCAGCCGGGTTCCGAGCAGGACATCGTGCTCGCAATCGACG
TGTCCGGGGGCATGTCGGGTCCGGCGCTGGACGACGTGAAGCGCG
CCGCATCGGATTTCGTGCGGCAGGCGCCGGCCGGCGCCCACATCG
GAATCGTCGCGATCTCGTCGACGCCACAGGTGCTCTCGGAACTGAC
GACGGACTCCGAGGACCTGCTCCGCAGGATCGACGGACTGAAGGCG
GGCGGCAACAGCGCGATCGCAGATTCGGTGGTGACCGCCGCCGAG
ATGCTCGAGCGCGGCGAAGCGGCCAACAACATCCTGCTTCTGTTGA
CGGACGGCGCCGACACGTCGAGTGCACACTCGATGTCGGAACTCCC
GTCCGTCCTGAGTCGGTCGCGCGCGTCGCTGTACGCCGTGCAGATG
TCGACACCCGAGACGAACTCTGCTCTCCTGCAGCAGGTTGCGCGGG
AGTCGCGCGGTCAGTACGCGTCTGCGGGTGATACGGCGGCGCTGG
GTGCGATCTACCAGTCGGCCGCTCGCGCGCTCGGAAACCTGTACGT
CGTCCGATACCGATCGGAAGCGAATGGCGATACCCAGGTGGTGGCG
AGCGTGCGCAGCGGCGCAGCCGGCCGAGTGAGCGATCCGTTCCCG
GTGACATTGCCCGGTGTGGTGCCGACGCCGAGCGTCGTCGCCGGG
ACCGTCGACGGTTTCTTCACGTCTTCGACGGGGCTGGTGATCGGGC
TCCTAGCGTGCTACTCGGCGCTTGCGGGAGGCGTGCTGGCGGTCGC
CGGTAGAGCGCCCGCGAGGATTTCGGCAGCACGTCGTGGGCGGCA
GGACGGACGGGACTCGATGCTGTCCCGATTCGCGGAACGGCTGGTG
CAGTGGATCGATCAGAACCTGAGGAGACGCGGACGCATCGCTGCCC
GCACCCAGGCGCTACAGGAGGCGGGGCTGAAGCTTCGTCCAGGTGA
CTTCATCGCCCTGGTCGGTGCTGCGGCGATCACCGCTGCGGCGATC
GGTCTCCTGGCTTCGGGCATCGTGGCGGCGCTCTTGCTCGCGGCGA
TCACAGTGGGATTGTCGAGAATCTATCTCCGTGTGATGGCCGGTAGG
CGTCGGGCCGCGTTCGCTGATCAGCTCGACGATTCCCTGCAGCTGC
TGGCCAGCAATCTCCGAGCCGGGCACAGCATGCTCCGAGCGCTCGA
TTCCCTTTCCCGAGAGGCGGAGGTGCCGACTTCGGAGGAGTTCGCT
CGGATCGTCAACGAGACTCGGGTGGGACGTGATCTCAACGAGTCTC
TCGACGACGTGGCCCGGCGGATGCGAAGTGACGATTTCAACTGGAT
AGCTCAGGCAATCGCCATCAACCGTGAGGTCGGAGGCGACCTCGCG
GAAGTCCTCGACCAGGTGGGCAACACCATTCGAGAGCGAAATCAGAT
TCGACGGCAGGTGAAAGCCCTTGCTGCCGAGGGGAAACTGTCCGCC
TACGTGCTGATGGCGCTGCCCTTCGGTCTCACCGCATTTCTGCTCGT
CTCGAATCCGGACTACCTGTCGAAGTTGACGGGTAGCGCCATCGGC
TACGTGATGATCGCGGTGGGGCTCGTCATGCTGACCGTCGGTGGGC
TGTGGATGAACAAGGTTGTCTCGGTCAAGTTCTAG SEQ ID NO 9 rpII REQ_18430>
- 1946262:1947152 GTGATTCCACCGCTGGTGCTCATGGCGGCGCTGTCCGTCGGCGGGG
CGTTGGGTGTTCTGGTGTGGTTGACGGTCGGCGCCCGAGATCCGGA
ACGCGGACCCGCCCTTCGGAACCTGCAGTCGCAGCTGGCGTTGCCG
ATTCCGGAGTCGGGAGGCGCGCCACCGCTTTCGCTCGGCCGATTCG
TGAAGCTGCTGTCGCCGCCCGGGACGATGGCCCGCTTGGAACGACT
GCACATCCTTGCCGGTCGTCCAGCGGCGTGGGTTCCGGAACGGGCC
GCGATGGCGAAGATCGTTCTCGCCGCGGCCGCCGCCCTGCTCGGC
CTTCTCGCGGTGGGTGCGTCGCCTGGCGTCGGCCGGGTGCTGTTCG
CTGCGGCCGCCGTCGCGCTGGCGTATTTCGTCCCGGAACTTCTCCT
GCAGAGCAGGGGGCAGGAGCGCCAAGCCGCGATCGAACTGGCGCT
TGCCGACACCCTCGACCAGATGACGATCGCAGTCGAGGCGGGCCTG
GGGTTCGAAGCCGCCATGCAGCGGGCCGCGAAGAACGGAAAGGGG
CCGCTGGCCGAGGAATTCATCCGGACATTGCAGGACATACAGATGG
GGCAGTCGAGGCGAATCGCGTACCTGGATCTTGCCGCCAGAACGAA
AGCACCCAACTTGCGGAGGTTCCTTCGGGCCGTCATCCAAGCCGAC
GAGTACGGCGTGGCCATCGCCGAGGTCCTGCGGACCCAGGCCTCG
GAGATGCGTCTGAAACGCCGTCAGAGTGCTGAGGAGAAGGCGATGA
AGGTTCCGGTGAAGGTGCTGTTTCCGTTGATGACCTGCATCCTGCCG
ACCATCTTCATCGTGATCCTGGGTCCGGCGGTGATCAACATGATGGA GGTCTTGGGCGGTATGTAA
SEQ ID NO 12 RpIA:
VIVAAGVGAALLGILAGAFANSAIDRVRLETACAEPKSTPTGSTPPPPSP
ASAVATRIAMIDTITRRHDISARRVLVELATALLFVGITLRLAALGLLPA
TPAYLWFAAVGIALAVIDIDCKRLPNFLVVPSYPIVFACLSVGSVVTGDW
SALLRAAIGAAVLFGVYFVLALIYPAGMGFGDVKLAGVIGAVLAYLSYGT
LLVGAFLAFLVAALVGLIILVTRRGRIGTTIPFGPYMIAAAIVAILAADP LARAYLDWAAAA SEQ
ID NO 13 RpIB: MNLFFANLYLMGLDVKDRLTRDDRGATAVEYGLMVAGIAMVIIVAVFAFG
DKITDLFDGFNFDDPGGE SEQ ID NO 14 RpIC:
MKRLTSDSGVAAVEFALVVPILITLVLGIVEFGRGYNVQNAVSAAAREGA
RTMAIKKDPAAARAAVKGAGVFSPAITDAEICISTSGTQGCSATSCPSGS
TVTLTVSYPLEYMTGLFPGKPTLTGTGVMRCGG SEQ ID NO 15 RpID:
MSNDERGVVAVLVAILMVVLLGCAAISVDIGANYVVKRQLQNGADAAALA
VAQESSCKAGSSASSVSSLVQANVNSSSAASAAVIDGVKRKVTVTASAV
GDDGLAGRRNVFAPVLGVDRSEISASATASCVFPLGGTAELPLTFHKCH
FDESRSLDVKILVAYNVTAPRCNGTSGNAAPGNFGWLQGANGRCPAKI
DAAVYATPGDTGNNIPGPCKDTIKQFQNAVVRVPIYDVAGGTGSGGWF
HVVGLAAFKIQGYRLSGNPEFNWNNDVHGALSCTGSCRGIIGTFVKIVSL
DSDLTPGGIDFGVSTISLLD SEQ ID NO 16 RpIE:
LRTRIIAAICAIVLAVAGTLALISYVRGADARALAGTRTVDVLVADQTIP
KNTPADSLVGMVVVKKLPEMAVLPDRVTSLDQLSGKVALTDLLPGEQLVS
ARFVDPATARSQDQGGIPEGMQEVTVLLEPQRALGGHIASGDTVGVFMSF
SPPVKNYETHLRLQKVRVTRVQGTFSNADEGDSATVDSSPSPAPTEAFL
VSLAVDVPMAERVVFAAEHGTIWLSNEPPSSNEAGASVVSP EGVFR SEQ ID NO 17 RpIF:
MSRIVLLTDRDDFARRVYHAADGNLLVLPAQPVPRGPAQLVGLGVTVQP
EVLVLGPDVPEVEGLSLAGRIDHSTPGTTVVLASDAGTDVWLRAMRAGV
RDVMSPEAEIADVRAVLDRAGQAALARRQGASAPAEQHAVQGKVIVVA
SPKGGTGKTTVATNLAVGLAAAAPHSTVLVDLDVQFGDVASALQLVPEH
CLTDAVAGPASQDMIVLKTVLTPHSTGLHALCGSDSPAAGDSITGEQVST
LLTQLAAEFRYVVVDTAPGLLEHTLAALDLATDVVLVSGMDVPSVRGMH
KELQLLTELNLGPVVRHVVLNFADRREGLTVQDIQNTIGVPADIVIKRSK
AVALSTNRGVPLLQNPGRDRTAKELWRLVGRIDPAPDTAKGGRARHRAA EAVGAK SEQ ID NO
18 RpIG: MRLSQRLEAVRGAAPVEAAAPIPPGKQGKAKTSLPPADALAELKDRASA
ALYTRIGTRFNDSSLSEEQLHLLVREELAEIVEQETTPLTFDERQRLLRE
VADEVLGHGPLQRLLEDPSVTEIMVNSHDMVYVERDGTLVRSSARFADEA
HLRRVIERIVSAVGRRIDESSPLVDARLADGSRVNAVIPPLAFNGSSLTI
RKFSKDPFQVDDLIAFGTLSHEMAELLDACVQARLNVIVSGGTGTGKTTL
LNVLSSFIPEGERIVTIEDAVELQLQQDHVVRLESRPPNIEGKGAVTIRD
LVRNSLRMRPDRIVVGECRGGESLDMLQAMNTGHDGSLSTVHANSPRDAI
ARLETLVLMAGMDLPLRAIREQIASAVDVIVQLTRLRDGTRRVTHVTEVQ
GMEGEIVTLQDAFLFDYSAGVDARGRFLGRPQPTGVRPRFTDRFRDLGI ALSPSVFGVGEPSRGRV
SEQ ID NO 19 RpIH:
MSRCVVAVVLALGAGVLGIPAVAAAAEEAVQVSAVDTTRFPDIEVSILAP
PGIEGQAIDPGTFALTEGGVPREIEVRQQPGSEQDIVLAIDVSGGMSGPA
LDDVKRAASDFVRQAPAGAHIGIVAISSTPQVLSELTTDSEDLLRRIDGL
KAGGNSAIADSVVTAAEMLERGEAANNILLLLTDGADTSSAHSMSELPSV
LSRSRASLYAVQMSTPETNSALLQQVARESRGQYASAGDTAALGAIYQSA
ARALGNLYVVRYRSEANGDTQVVASVRSGAAGRVSDPFPVTLPGVVPT
PSVVAGTVDGFFTSSTGLVIGLLACYSALAGGVLAVAGRAPARISAARRG
RQDGRDSMLSRFAERLVQWIDQNLRRRGRIAARTQALQEAGLKLRPGD
FIALVGAAAITAAAIGLLASGIVAALLLAAITVGLSRIYLRVMAGRRRAA
FADQLDDSLQLLASNLRAGHSMLRALDSLSREAEVPTSEEFARIVNETRV
GRDLNESLDDVARRMRSDDFNWIAQAIAINREVGGDLAEVLDQVGNTIRE
RNQIRRQVKALAAEGKLSAYVLMALPFGLTAFLLVSNPDYLSKLTGSAIG
YVMIAVGLVMLTVGGLWMNKVVSVKF SEQ ID NO 20 RpII:
VIPPLVLMAALSVGGALGVLVWLTVGARDPERGPALRNLQSQLALPIPES
GGAPPLSLGRFVKLLSPPGTMARLERLHILAGRPAAWVPERAAMAKIVLA
AAAALLGLLAVGASPGVGRVLFAAAAVALAYFVPELLLQSRGQERQAAIE
LALADTLDQMTIAVEAGLGFEAAMQRAAKNGKGPLAEEFIRTLQDIQMG
QSRRIAYLDLAARTKAPNLRRFLRAVIQADEYGVAIAEVLRTQASEMRLK
RRQSAEEKAMKVPVKVLFPLMTCILPTIFIVILGPAVINMMEVLGGM
[0092] Preferred features and embodiments of each aspect of the
invention are as for each of the other aspects mutatis mutandis
unless context demands otherwise.
[0093] Each document, reference, patent application or patent cited
in this text is expressly incorporated herein in their entirety by
reference, which means it should be read and considered by the
reader as part of this text. That the document, reference, patent
application or patent cited in the text is not repeated in this
text is merely for reasons of conciseness. Reference to cited
material or information contained in the text should not be
understood as a concession that the material or information was
part of the common general knowledge or was known in any
country.
[0094] Throughout the specification, unless the context demands
otherwise, the terms `comprise` or `include`, or variations such as
`comprises` or `comprising`, `includes` or `including` will be
understood to imply the includes of a stated integer or group of
integers, but not the exclusion of any other integer or group of
integers.
[0095] By "consisting essentially of" it is meant that a nucleic
acid does not include additional, substituted or deleted
nucleotide(s) to a polynucleotide sequence of the invention
described herein or a polypeptide does not include additional,
substituted, or deleted amino acids which significantly alter the
character of a sequence of the invention such that it is not
immunogenic and biologically active.
[0096] As used herein, the singular forms "a", "an", and "the"
include the corresponding plural reference unless the context
clearly dictates otherwise.
[0097] Where a range of values is expressed, it will be understood
that this range encompasses the upper and lower limits of the range
and all values in between these limits.
[0098] The terms "polypeptide", "protein" and "peptide" are herein
used interchangeably.
[0099] The term "isolated" refers to materials, such as nucleic
acid molecules, which are substantially free or otherwise removed
from components that normally accompany or interact with the
materials in a naturally occurring environment. An isolated nucleic
acid typically contains less than about 50%, preferably less than
about 75%, and most preferably less than about 90% of the
components with which it was originally associated. Polypeptides,
antibodies and nucleic acids of the invention as disclosed herein
can be isolated.
[0100] The terms "polynucleotide", "polynucleotide sequence", and
"nucleic acid sequence" are used interchangeably herein. A
"polynucleotide" as used herein refers to purine- and
pyrimidine-containing polymers of any length, either
polyribonucleotides or polydeoxyribonucleotides, which can be
single or double stranded, such as, for example, DNA-DNA, DNA-RNA
and RNA-RNA. A polynucleotide may optionally contain synthetic,
non-natural or altered nucleotide bases. A polynucleotide in the
form of a polymer of DNA may be comprised of one or more strands of
cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
[0101] A "derivative" of a polypeptide as used herein will be
understood to include polypeptides which have been subject to
chemical modifications, including esterification, amidation,
reduction, methylation, fusion to another peptide and the like. The
polypeptide derivatives may be modified such that the modifications
increase the stability and/or immunogenicity and/or bioavailability
of the polypeptide derivative in comparison to the unmodified
polypeptide. Covalent derivatives of the peptides or polypeptides
of the invention can be prepared by linking the chemical moieties
to functional groups on the amino acid side chains or at the
N-terminus or C-terminus of the antigenic polypeptide. Conjugation
of a polypeptide to another peptide may further be achieved by
genetic means through the use of recombinant DNA techniques that
are well know in the art, such as those set forth in the teachings
of Sambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed.
Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory Press, (1989)
and F.M. Ausubel et al. Current Protocols in Molecular Biology,
Eds. J.Wiley Press (2006), the relevant portions of which are
incorporated herein by reference.
[0102] A "variant" polypeptide of the invention can be a
polypeptide which has an amino acid sequence which differs from the
polypeptide encoded by SEQ ID NO 1, 2, 3, 4, 5, 6, 7, 8 or 9 due to
the presence of one or more deletions, insertions, or substitutions
of amino acid residues. In embodiments, a variant has at least 85%,
86%, 87%, 88%, 89%, preferably at least 90%, 91%, 92%, 93%, 94%,
and more preferably 95%, 96%, 97%, 98%, 99% but less than 100%
contiguous amino acid sequence identity to the corresponding
polypeptide encoded by the nucleotide sequence as disclosed herein.
Percentage identity may be determined using, for example computer
programs as would be known by one skilled in the art.
[0103] Variants can include polypeptides in which individual amino
acids of the polypeptide of the invention are substituted by other
amino acids which are closely related as understood in the art, for
example, substitution of one hydrophobic residue such as
isoleucine, valine, leucine or methionine for another, or the
substitution of one polar residue for another, such as arginine for
lysine, glutamic for aspartic acid or glutamine for asparagine.
[0104] In embodiments, a fragment of a polypeptide of the present
invention can consist of a truncated version of a polypeptide of
the invention which has been truncated by 1, 2, 3, 4 or more than
5, more than 10, or more than 20 amino acids. An antigenic fragment
may be generated using for example C-terminal deletion of any one
of the polynucleotide sequences of the genes as listed in Table 1
or Table 2 and said C-terminal deletion constructs may then be
inserted into a suitable prokaryotic or eukaryotic expression
plasmid. The antigenic activity of the expression products derived
from the constructs may then be tested by assessing reactivity with
antisera from naturally and/or experimentally infected horse or
foals using immunoblotting methods. Alternatively a series of
synthetic polypeptide fragments with greater than 85%, greater than
90%, greater than 95%, or 100% sequence identity to portions of any
one the polypeptides encoded by a polynucleotide sequence of a gene
of Table 1 or more preferably Table 2 can be generated. These
peptides may then be reacted with antisera from naturally or
experimentally infected horses using an ELISA method to determine
which peptide fragments are antigenic. Alternatively, synthetic
peptides may be used to immunise, for example, mice, rabbits, or
horses and the antisera produced can be assessed for reactivity
with R. equi using indirect immunofluorescence assays. In this way
immunogenic fragments may be identified and R. equi-specific
antisera may be produced. These two latter approaches described are
particularly advantageous for small peptides that contain linear,
continuous epitopes.
[0105] "Operably linked" means that a nucleic acid molecule is
placed in functional relationship with another nucleic acid
molecule. Generally an operably linked promoter will be linked such
that it is contiguous with and in the same reading phase as the
gene to be expressed.
[0106] Generally the terms "treating", "treatment" and the like are
used to mean affecting a subject tissue or cell to obtain a desired
pharmacological and/or physiological effect. As used herein, the
term "treatment" and associated terms such as "treat" and
"treating" means the reduction of the progression, severity and/or
duration of infection or for the amelioration of at least one of
the symptoms thereof by R. equi or may be prophylactic
(preventative treatment). The term `treatment` therefore refers to
any regimen that can benefit a subject. References herein to
"therapeutic" and "prophylactic" treatments are to be considered in
their broadest context. The term "therapeutic" does not necessarily
imply that a subject is treated until total recovery. Similarly,
"prophylactic" does not necessarily mean that the subject will not
eventually contract a disease condition.
[0107] As used herein, the term "subject" refers to an animal,
preferably a mammal and in particular a horse.
FIGURES
[0108] Embodiments of the present invention will now be described
by way of example only with reference to the accompanying figures
in which:
[0109] FIG. 1 illustrates the R. equi pilus locus (rpl). (A) The 9
Kb rpl horizontally acquired (HGT) island (REQ18350-430) is absent
from nonpathogenic Rhodococcus spp. (e.g. R. jostii RHA1 and R.
erythropolis PR4). rpl genes have were detected in all R. equi
clinical isolates (.apprxeq.300 isolates tested). rpl gene products
which are considered to be encoded are: A, prepilin peptidase; B,
pilin subunit; C, TadE minor pilin; D, putative lipoprotein; E,
CpaB pilus assembly protein; F, CpaE pilus assembly protein; GHI,
Tad transport machinery. (B) Electron micrograph of R. equi 103S
pili (indicated by arrowheads). Bar=0.5 .mu.m. (C) R. equi pili
visualized by immunofluorescence microscopy (.times.1,000
magnification). Reproduced from Letek et al. 2010, PLoS Genet. 6:
e1001145).
[0110] FIG. 2 illustrates a demonstration by targeted mutant
construction and genetic re-complementation analysis that the rpl
locus encodes the R. equi pilus. Negative staining transmission
electron micrographs of wild-type R. equi 103S (WT) (panel A),
isogenic rplB deletion mutant of 103S (.DELTA.rplB, apiliated)
(panel B), rplB-complemented mutant (piliated) (panel C), and
mock-complemented mutant with an empty vector (no rplB gene).
Bar=0.5 .mu.m (panel D).
[0111] FIG. 3 illustrates the effect of rplB gene deletion and
complementation on R. equi adhesion to (A) macrophages (J774A.1
cell line) and (B) epithelial cells (HeLa cell line), two key
target cell types in the pathogenesis of airborne lung infection.
Data expressed as percentage of the control (WT); mean of at least
three independent duplicate experiments.+-.SEM.
[0112] FIG. 4 illustrates the adhesion phenotype to (A) epithelial
cells (HeLa cell line) and (B) macrophages (J774A.1 cell line) with
additional rpl knock-out mutants (rplA and rplE).
[0113] FIG. 5 illustrates Rpl pili are essential for R. equi lung
colonization in mice as demonstrated using a novel in vivo lung
infection model in mice developed by the inventors. It is based on
a competitive virulence assay in which each mouse receives an
intranasal inoculum containing 50% of wild-type (WT) R. equi
bacteria and 50% of mutant (.DELTA.rplB) R. equi bacteria. t=0
means 60 min after delivery of the intranasal inoculum.
[0114] FIG. 6 illustrates production in rabbits of a specific
antibody against the putative R. equi pilin subunit (RplB). (A)
Amino acid sequence of putative RplB prepilin and of the C-terminal
peptide used to raise a rabbit polyclonal antibody (boxed).
Arrowhead indicates putative cleavage site of the prepilin. (B)
Immunodetection of the RplB pilin by SDS-PAGE western blot analysis
of whole cell extracts of wild-type R. equi (WT), an isogenic
in-frame deletion rplB mutant (.DELTA.rplB), the rplB-complemented
mutant (.DELTA.rplB+rplB), and a mock-complement mutant
(.DELTA.rplB+vector), using the anti-RplB peptide antibody (diluted
1:1,000; secondary antibody, alkaline phosphatase-conjugated mouse
anti-rabbit monoclonal antibody, 1:10,000 diluted; reaction
revealed with NBT/BCIP substrate. The anti-Rpl antibody
specifically detects the Rpl pilin subunit in WT and re-completed
rpl mutant, not in the apiliated rpl mutant and mock-complemented
mutant. (C) Detection of Rpl pili production in R. equi by
immunofluorescence using the anti-RplB peptide antibody and the
same bacteria as in (B) (630.times. magnification, Leica AF6000
microscope).
[0115] FIG. 7 illustrates Inhibition of R. equi attachment to (A)
macrophages and (B) epithelial cells by an anti-RplB antibody.
Prior to the adhesion assay, the antibody raised against the RplB
(pilin subunit) peptide (see FIG. 6A) was incubated for 60 min at
37.degree. C. (40 .mu.l/ml of a suspension in cell culture medium
of exponentially grown R. equi bacteria at a density calculated for
a multiplicity of infection of 15:1). As a control, the R. equi
bacterial cell suspension was pre-incubated with an irrelevant
antiserum (anti-Listeria monocytogenes rabbit polyclonal
antibody).
[0116] FIG. 8 illustrates RplB pilin antigens are recognized in
vivo and elicit a strong antibody response in naturally infected
foals. Representative example of the reactivity against the Rpl
pilin of horse sera from bacteriologically confirmed cases of foal
pneumonia, as determined by SDS-PAGE western blot analysis with
whole cell extracts of wild-type R. equi (WT) and the isogenic
.DELTA.rplB mutant. All convalescent sera tested to date gave a
strong reaction against the RplB pilin antigen whereas normal
(non-case) sera did not. The Rpl pili dissociate into 18 kDa
polypeptides that probably correspond to SDS-resistant
homo-tetramers (predicted molecular mass of RplB pilin, 4.95 kDa)
that remain non-covalently bound by strong monomer-monomer
interactions via the N-terminal hydrophobic region of the pilin
subunit. (A) indicates RplB is the first antigen detected in a
curde R. equi protein preparation by the antibodies present in case
sera.
[0117] FIG. 9 illustrates variability of RplB amino acid sequence
in R. equi strains and of other Rpl proteins.
[0118] FIG. 10 illustrates the nucleotide sequences encoding Rpl
proteins of other strains of R. equi.
DETAILED DESCRIPTION OF THE INVENTION
[0119] As indicated above, the inventors have identified
polypeptides which play an important role in virulence of R equi
and have used this knowledge to identify polypeptides which can be
used to mediate an immune response in infected subjects,
particularly horses, and in particular foals. Whilst the amino acid
sequences of the polypeptides determined for the identified strain
are noted, as will be understood, biologically active immunogenic
fragments, derivatives or variants of such a polypeptide can also
be used. As discussed variant polypeptides can comprise amino acid
percent identity with the amino acid sequences disclosed herein.
Alternatively, polypeptides of the invention may be encoded by
variant nucleic acid sequences which have nucleotide percent
identity with the polynucleotide sequences disclosed herein.
[0120] The percent identity of two or more sequences may be
determined by visual inspection and mathematical calculation.
Alternatively, the percent identity of two nucleic acid sequences
can be determined by comparing sequence information using the GAP
computer program, version 6.0 described by Devereux et al. (Nucl.
Acids Res. 12:387, 1984) and available from the University of
Wisconsin Genetics Computer Group (UWGCG). The preferred default
parameters for the GAP program include: (1) a unary comparison
matrix (containing a value of 1 for identities and 0 for
non-identities) for nucleotides, and the weighted comparison matrix
of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as
described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence
and Structure, National Biomedical Research Foundation, pp.
353-358, 1979; (2) a penalty of 3.0 for each gap and an additional
0.10 penalty for each symbol in each gap; and (3) no penalty for
end gaps. Other programs used by one skilled in the art of sequence
comparison may also be used.
[0121] Polypeptides of the invention may be prepared by any of a
number of conventional techniques. A nucleic acid encoding a
peptide or a biologically active immunogenic fragment, derivative,
or variant thereof, may be subcloned into an expression vector for
production of the polypeptide or fragment. The DNA sequence
advantageously is fused to a sequence encoding a suitable leader or
signal peptide and/or a promoter operable in a cell into which the
nucleic acid is to be introduced. Alternatively, the desired
fragment may be chemically synthesized using known techniques. DNA
fragments also may be produced by restriction endonuclease
digestion of a full length cloned DNA sequence, and isolated by
electrophoresis on agarose gels. If necessary, oligonucleotides
that reconstruct the 5' or 3' terminus to a desired point may be
ligated to a DNA fragment generated by restriction enzyme
digestion. Such oligonucleotides may additionally contain a
restriction endonuclease cleavage site upstream of the desired
coding sequence, and position an initiation codon (ATG) at the
N-terminus of the coding sequence.
[0122] Polymerase chain reaction (PCR) procedure also may be
employed to isolate and amplify a DNA sequence encoding a desired
polypeptide fragment. Oligonucleotides that define the desired
termini of the DNA fragment are employed as 5' and 3' primers. The
oligonucleotides may additionally contain recognition sites for
restriction endonucleases, to facilitate insertion of the amplified
DNA fragment into an expression vector. PCR techniques are
described in Saiki et al., Science 239:487 (1988); Recombinant DNA
Methodology, Wu et al., eds., Academic Press, Inc., San Diego
(1989), pp. 189-196; and PCR Protocols: A Guide to Methods and
Applications, Innis et al., eds., Academic Press, Inc. (1990).
[0123] The invention encompasses polypeptides and biologically
active immunogenic fragments, derivatives, or variants thereof in
various forms, including those that are naturally occurring or
produced through various techniques such as procedures involving
recombinant DNA technology. For example, nucleotides encoding
polypeptides of the invention can be derived from SEQ ID NO 1, 2,
3, 4, 5, 6, 7, 8, or 9 by in vitro mutagenesis, which includes
site-directed mutagenesis, random mutagenesis, and in vitro nucleic
acid synthesis. Such forms include, but are not limited to,
derivatives, variants, and oligomers, as well as fusion proteins or
fragments thereof.
Polypeptide Derivatives
[0124] Embodiments of a derivative of a polypeptide of the
invention can comprise one or more non-naturally occurring amino
acids or amino acid analogs, including non-genetically encoded
L-amino acids, synthetic L-amino acids or D-enantiomers of an amino
acid. Suitably, embodiments of a derivative can comprise one or
more residues selected from the group consisting of:
hydroxyproline, .beta.-alanine, 2,3-diaminopropionic acid,
.alpha.-aminoisobutyric acid, N-methylglycine (sarcosine),
ornithine, citrulline, t-butylalanine, t-butylglycine,
N-methylisoleucine, phenylglycine, cyclohexylalanine, norleucine,
naphthylalanine, pyridylananine 3-benzothienyl alanine
4-chlorophenylalanine, 2-fluorophenylalanine,
3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine,
1,2,3,4-tetrahydrotic isoquinoline-3-carboxylic acid
.beta.-2-thienylalanine, methionine sulfoxide, homoarginine,
N-acetyl lysine, 2,4-diamino butyric acid, p-aminophenylalanine,
N-methylvaline, homocysteine, homoserine, .epsilon.-amino hexanoic
acid, .delta.-amino valeric acid, 2,3-diaminobutyric acid and
mixtures thereof. Other amino acid residues that are useful for
making the polypeptides and polypeptide derivatives described
herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook
of Biochemistry and Molecular Biology, CRC Press, Inc., and the
references cited therein.
[0125] In embodiments, derivatives of polypeptides of the invention
can also comprise an isostere of a polypeptide. The term "isostere"
as used herein is intended to include a chemical structure that can
be substituted for a second chemical structure because the steric
conformation of the first structure fits a binding site specific
for the second structure. The term specifically includes peptide
back-bone modifications (i.e., amide bond mimetics) known to those
skilled in the art. Such modifications include modifications of the
amide nitrogen, the .alpha.-carbon, amide carbonyl, complete
replacement of the amide bond, extensions, deletions or backbone
crosslinks. Several peptide backbone modifications are known,
including .psi.[CH2S], .psi.[CH2NH], .psi.[CSNH2], .psi.[NHCO],
.psi.[COCH2], and .psi.[(E) or (Z) CH.dbd.CH]. In the nomenclature
used above, .psi. indicates the absence of an amide bond. The
structure that replaces the amide group is specified within the
brackets. Other modifications include, for example, an N-alkyl (or
aryl) substitution (.psi.[CONR]), or backbone crosslinking to
construct lactams and other cyclic structures. In another example,
a polypeptide derivative may be a retro-peptide analog. A
retro-peptide analog comprises a reversed amino acid sequence of a
polypeptide described herein. For example, a retro-peptide analog
of a polypeptide comprises a reversed amino acid sequence of a
sequence set forth in any one of SEQ ID NO 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20. Retro-inverso polypeptides may be complete
or partial. Complete retro-inverso peptides are those in which a
complete sequence of a polypeptide described herein is reversed and
the chirality of each amino acid in a sequence is inverted, other
than glycine, because glycine does not have a chiral analog.
Partial retro-inverso polypeptides are those in which only some of
the peptide bonds are reversed and the chirality of only those
amino acid residues in the reversed portion is inverted. For
example, one or two or three or four or five or more than 10, more
than 20, more than 30, more than 40 or more than 50 amino acid
residues are D-amino acids. Suitably a polypeptide of and for use
in the present invention may be further modified using at least one
of C and/or N-terminal capping, and/or cysteine residue capping.
Suitably, a polypeptide of and for use in the present invention may
be capped at the N terminal residue with an acetyl group. Suitably,
a polypeptide of and for use in the present invention may be capped
at the C terminal with an amide group. Suitably, thiol groups of
cysteines of polypeptides of the invention may be capped with
acetamido methyl groups. In embodiments, the term derivative can
include scrambled polypeptides comprising immunodominant epitopes
of the rpl encoded pilus for example fragments of SEQ ID NOs 10,
11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In embodiments
derivatives can be encoded by rpl genes or fragments thereof which
encode immunodominant epitopes of Rpl pilus provided in tandem, or
as longer repeat stretches, for example concatemerized, to increase
the immunogenicity of the encoded polypeptides. In embodiments,
combinations of polypeptides of the invention (and corresponding
nucleic acid sequences) can be fused in a single polypeptide.
Polypeptide Synthesis
[0126] A polypeptide or a biologically active immunogenic fragment,
derivative, or variant thereof may be synthesized using any
suitable chemical method known to the person skilled in the art.
For example, synthetic peptides can be prepared using known
techniques of solid phase, liquid phase, or peptide condensation,
or any combination thereof, and can include natural and/or
unnatural amino acids. Amino acids used for peptide synthesis may
be standard Boc (N.alpha.-amino protected
N.alpha.-t-butyloxycarbonyl) amino acid resin with the
deprotecting, neutralization, coupling and wash protocols of the
original solid phase procedure of Merrifield, J. Am. Chem. Soc.,
85:2149-2154, 1963, or the base-labile Na-amino protected
9-fluorenylmethoxycarbonyl (Fmoc) amino acids described by Carpino
and Han, J. Org. Chem., 37:3403-3409, 1972. Both Fmoc and Boc
N.alpha.-amino protected amino acids can be obtained from various
commercial sources, such as, for example, Fluka, Bachem, Advanced
Chemtech, Sigma, Cambridge Research Biochemical, Bachem, or
Peninsula Labs.
[0127] Generally, chemical synthesis methods comprise the
sequential addition of one or more amino acids to a growing peptide
chain. Normally, either the amino or carboxyl group of the first
amino acid is protected by a suitable protecting group. The
protected or derivatized amino acid can then be either attached to
an inert solid support or utilized in solution by adding the next
amino acid in the sequence having the complementary (amino or
carboxyl) group suitably protected, under conditions that allow for
the formation of an amide linkage. The protecting group is then
removed from the newly added amino acid residue and the next amino
acid (suitably protected) is then added, and so forth. After the
desired amino acids have been linked in the proper sequence, any
remaining protecting groups (and any solid support, if solid phase
synthesis techniques are used) are removed sequentially or
concurrently, to render the final polypeptide. By simple
modification of this general procedure, it is possible to add more
than one amino acid at a time to a growing chain, for example, by
coupling (under conditions which do notracemize chiral centers) a
protected tripeptide with a properly protected dipeptide to form,
after deprotection, a pentapeptide. See, e.g., J. M. Stewart and J.
D. Young, Solid Phase Peptide Synthesis (Pierce Chemical Co.,
Rockford, Ill. 1984) and G. Barany and R. B. Merrifield, The
Peptides: Analysis, Synthesis, Biology, editors E. Gross and J.
Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254,
for solid phase peptide synthesis techniques; and M. Bodansky,
Principles of Peptide Synthesis, (Springer-Verlag, Berlin 1984)and
E. Gross and J. Meienhofer, Eds., The Peptides: Analysis.
Synthesis. Biology, Vol. 1, for classical solution synthesis.
Typical protecting groups include t-butyloxycarbonyl (Boc),
9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz);
p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl);
biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl,
isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl,
isopropyl, acetyl, o-nitrophenylsulfonyl and the like.
[0128] Typical solid supports are cross-linked polymeric supports.
These can include divinylbenzene cross-linked-styrene-based
polymers, for example, divinylbenzene-hydroxymethylstyrene
copolymers, divinylbenzene-chloromethylstyrene copolymers and
divinylbenzene-benzhydrylaminopolystyrene copolymers.
[0129] A peptide or a biologically active immunogenic fragment,
derivative, or variant thereof as described herein according to any
embodiment can also be chemically prepared by other methods such as
by the method of simultaneous multiple peptide synthesis. See, e.
g., Houghten Proc. Natl. Acad. Sci. USA 82: 5131-5135, 1985 or U.S.
Pat. No. 4,631,211.
Recombinant Polypeptide Production
[0130] Alternatively, or in addition, a peptide or a biologically
active immunogenic fragment, derivative, or variant thereof can be
produced as a recombinant protein. To facilitate the production of
a recombinant polypeptide, nucleic acid encoding the same is
preferably isolated or synthesized. Typically the nucleic acid
encoding the recombinant protein is/are isolated using a known
method, such as, for example, amplification (e.g., using PCR or
splice overlap extension) or isolated from nucleic acid from R.
equi using one or more restriction enzymes or isolated from a
library of nucleic acids.
[0131] Methods for such isolation will be apparent to the ordinary
skilled artisan and/or described in Ausubel et al (In: Current
Protocols in Molecular Biology. Wiley Interscience, ISBN 047
150338, 1987), Sambrook et al (In: Molecular Cloning: Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New
York, Third Edition 2001).
[0132] For expressing protein by recombinant means, a
protein-encoding nucleic acid is placed in operable connection with
a promoter or other regulatory sequence capable of regulating
expression in a cell-free system or cellular system. For example,
nucleic acid comprising a sequence that encodes a polypeptide of
the pili of R. equi is placed in operable connection with a
suitable promoter and maintained in a suitable cell for a time and
under conditions sufficient for expression to occur.
[0133] A number of other gene construct systems for expressing a
nucleic acid of a gene selected from Table 1 or Table 2 in
bacterial cells are well-known in the art and are described for
example, in Ausubel et al (In: Current Protocols in Molecular
Biology. Wiley Interscience, ISBN 047 150338, 1987), and Sambrook
et al (In: Molecular Cloning: Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, New York, Third Edition
2001).
[0134] A wide range of additional host/vector systems suitable for
expressing a polypeptide of the present invention are available
publicly, and described, for example, in Sambrook et al (In:
Molecular cloning, A laboratory manual, second edition, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y., 1989).
[0135] Following expression of a polypeptide, isolation and
purification of the polypeptide may be accomplished by any suitable
technique, as would be known in the art.
Compositions
[0136] A polypeptide or a biologically active immunogenic fragment,
derivative, or variant thereof may be administered alone, but will
preferably be administered as a pharmaceutical composition, which
will generally comprise a suitable pharmaceutically acceptable
excipient, diluent or carrier selected depending on the intended
route of administration. Examples of suitable pharmaceutical
carriers include; water, glycerol and ethanol.
[0137] The term "carrier or excipient" as used herein, refers to a
carrier or excipient that is conventionally used in the art to
facilitate the storage, administration, and/or the biological
activity of an active compound. A carrier may also reduce any
undesirable side effects of the active compound. A suitable carrier
is, for example, stable, e.g., incapable of reacting with other
ingredients in the formulation. In one example, the carrier does
not produce significant local or systemic adverse effect in
recipients at the dosages and concentrations employed for
treatment. Such carriers and excipients are generally known in the
art. Suitable carriers for this invention include those
conventionally used, e.g., water, saline, aqueous dextrose, and
glycols are preferred liquid carriers, particularly (when isotonic)
for solutions. Suitable pharmaceutical carriers and excipients
include starch, cellulose, glucose, lactose, sucrose, gelatin,
malt, rice, flour, chalk, silica gel, magnesium stearate, sodium
stearate, glycerol monostearate, sodium chloride, glycerol,
propylene glycol, water, ethanol, and the like.
[0138] Pharmaceutical composition adapted for oral administration
may be presented as discrete units such as capsules, soft gels, or
tablets; powders or granules; solutions or suspensions in aqueous
or non-aqueous liquids; edible foams or whips; or oil-in-water
liquid emulsions or water-in-oil liquid emulsions.
[0139] Pharmaceutical compositions provided as formulations adapted
for parenteral administration include aqueous and non-aqueous
sterile injection solutions which contain a polypeptide or a
biologically active immunogenic fragment, derivative, or variant
thereof or a antibody of the invention and optionally, buffers,
bacteriostats and solutes which render the formulation isotonic
with the blood of the intended recipient; and aqueous and
non-aqueous sterile suspensions which may include suspending agents
and thickening agents. The formulations may be presented in
unit-dose or multi-dose containers, for example sealed ampules and
vials, and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for
example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules and tablets.
Administration
[0140] As will be appreciated by a person of skill in the art,
selecting an administration regimen for a therapeutic composition
or vaccine of the invention depends on several factors, including
the serum or tissue turnover rate of a polypeptide of the invention
or an antibody invention, the level of symptoms, the immunogenicity
of the polypeptide, and the accessibility of the target cells in
the biological matrix. Preferably, an administration regimen
maximizes the amount of therapeutic compound delivered to the
subject consistent with an acceptable level of side effects.
Accordingly, the amount of polypeptide, antibody or composition
delivered depends in part on the polypeptide, antibody or
composition and the severity of the condition being treated.
[0141] A polypeptide or antibody can be provided, for example, by
continuous infusion, or by doses at intervals of, e.g., one day,
one week, or 1-7 times per week. A preferred dose protocol is one
involving the maximal dose or dose frequency that avoids
significant undesirable side effects. A total weekly dose depends
on the type and activity of the compound being used. Determination
of the appropriate dose is made by a veterinarian or clinician, for
example using parameters or factors known or suspected in the art
to affect treatment or predicted to affect treatment.
EXAMPLES
Example 1
[0142] Using electron microscopy and other microscopical techniques
we demonstrated that R. equi produces long, thick and apparently
rigid pili appendages, typically between two and four per bacteria
cell (FIG. 1 panels BC).
Example 2
Genome Sequencing
[0143] Genome sequencing of the complete genome sequence of R. equi
strain 103S was determined in an international collaborative
venture. The genome has just over 5 million base pairs and encodes
4598 genes of average length value 1009 pairs of nucleotides.
Example 3
[0144] Demonstration that the rpl (R. equi pili) locus (nucleotide
positions 1,938,280 to 1,947,152, locus tags REQ18350-430) encodes
the R. equi pilus by targeted mutant construction and genetic
re-complementation analysis.
[0145] An in-frame deletion mutant was constructed in the rplB gene
putatively encoding the Rpl pilin subunit (RplB). Homologous
recombination methodology previously devised (Navas et al. 2001,
Identification and mutagenesis by allelic exchange of choE,
encoding a cholesterol oxidase from the intracellular pathogen
Rhodococcus equi. J. Bacteriol. 183: 4796-4805), and a novel
suicide vector, pSelAct, for positive selection of double
recombinants on 5-fluorocytosine (5-FC) (van der Geize et al. 2008,
A novel method to generate unmarked gene deletions in the
intracellular pathogen Rhodococcus equi using 5-fluorocytosine
conditional lethality. Nucleic Acids Res. 36: el 51) was used in
this approach. The .DELTA.rplB mutant was complemented by stably
inserting the rplB gene plus corresponding promoter region into the
R. equi chromosome using the integrative vector pSET152 (Hong and
Hondalus 2008, Site-specific integration of Streptomyces PhiC31
integrase-based vectors in the chromosome of Rhodococcus equi. FEMS
Microbiol. Lett. 287: 63-68). As shown in FIG. 2, the inactivation
of the rplB gene results in loss of pili formation by R. equi. Pili
formation is restored upon reintroduction of the rplB gene in the
.DELTA.rplB mutant but not by complementation with an empty vector,
demonstrating that rplB is a gene directly responsible for the
piliated phenotype.
Example 4
[0146] Demonstration that the R. equi pili mediate attachment to
mammalian cells.
[0147] The .DELTA.rplB mutant was tested in adhesion assays using
monolayers of two cell types relevant to R. equi infection:
epithelial cells to which the pathogen have to adhere to during the
initial stages of lung colonization, and macrophages, which are
used as host cells for bacterial intracellular replication. The
rplB mutant was severely impaired in attachment to both eukaryotic
cell types, and its complementation with the rplB gene but not an
empty vector restored wild-type cytoadhesiveness (FIG. 3).
[0148] Two additional mutants were constructed in rplA and rplE
(FIG. 1A) and they also caused a significant reduction of R. equi
cytoadhesiveness (FIG. 4), indicating that other genes from the rpl
locus are involved in pilus-mediated attachment to eukaryotic cells
(not shown).
Example 5
[0149] Demonstration that the R. equi pili are essential for lung
colonization in vivo in a mouse model of R. equi infection.
[0150] A novel in vivo model of competitive R. equi lung infection
in mice was developed and used to test the virulence of the rplB
mutant in comparison to rplB-proficient (wild-type) bacteria. R.
equi wild-type and an isogenic rplB knock-out mutant in equal
amounts were inoculated intranasally to Balb/c mice. At specific
time points after infection, the bacterial population was
determined in lungs and tracheas to assess airway colonisation. The
spleens were also analysed to determine the capacity of the
bacteria to overwhelm local defences and spread deeper into host
tissues. FIG. 4 shows that the mutant, initially accounting for 50%
of the inoculum, was only detectable--in much less
proportion--during the two first time points sampled (0 and 24 hour
post inoculation), indicating that apiliated bacteria are
immediately cleared from the lungs and thus substantially less
virulent. In the first time point, only a very small fraction of
the bacteria that translocated to the spleen were mutants. These
data indicate that a wild-type capacity to attach to host cells via
the Rpl pili is essential for host colonisation by R. equi.
Example 6
[0151] Demonstration that the RplB (putative pilin subunit) protein
is antigenic in vivo in rabbits.
[0152] The synthetic RplB peptide indicated in FIG. 6A was used to
hyperimmunize rabbits. The antiserum specifically detected the RplB
pilin subunit in whole cell extracts of R. equi (FIG. 6B) and the
production of Rpl pili in R. equi by immunofluorescence (FIG. 6C),
indicating that it is immunogenic in vivo in rabbits.
Example 7
[0153] Demonstration that RplB elicits neutralizing antibodies that
inhibit R. equi attachment.
[0154] The rabbit hyperimmune anti-RplB antiserum was used in
attachment-inhibition assays in HeLa epithelial cells and J774A.1
macrophages. FIG. 7 shows that the RplB antiserum, but not an
irrelevant antiserum, inhibited R. equi cytoadhesion. Given the key
role of the Rpl pili in lung colonization by R. equi (FIG. 4),
these data indicate that RplB is a vaccine target to prevent lung
infection by the pathogen.
[0155] This is evidence that indicates that the pilin subunit RplB
is recognised by the immune system in vivo and the animal body
mounts a specific immune response with production of specific
antibodies to the R. equi pilin subunit RplB. As the polyclonal
antiserum containing anti-RplB antibodies inhibits attachment of R.
equi to monolayers of HeLa epithelial cells or J774 macrophages if
added to the infection assays, which effect is not seen if the Rpl
antiserum is not added, or if an unrelated control antiserum raised
against other bacteria (e.g. Listeria) is used, this indicates a
protective function of the antibodies through inhibition of
bacterial attachment to host cells, the first phase of host
colonisation during infection.
Example 8
[0156] Demonstration that the RplB putative pilin subunit is an
immunodominant antigen in naturally infected foals.
[0157] Using SDS-PAGE western immunoblotting and whole-cell
extracts from wild-type and rplB (apiliated) deletion mutant
bacteria, it was shown that the sera from natural cases of R. equi
infection in foals contain antibodies to the RplB putative pilin
subunit (FIG. 8). The RplB protein is the first detected in the
crude R. equi protein preparation by the antibodies present in the
case sera. Thus, the RplB pilin subunit is recognized in vivo by
the foal's immune system during R. equi infection and is an
immunodominant antigen. Normal, non-case sera did not react against
the RplB protein, indicating that this antigen provides a suitable
maker for the early detection and diagnosis of R. equi infection in
foals.
[0158] Although the invention has been particularly shown and
described with reference to particular examples, it will be
understood by those skilled in the art that various changes in the
form and details may be made therein without departing from the
scope of the present invention.
Sequence CWU 1
1
731702DNARhodococcus equi 1gtgatcgtcg cagcgggcgt cggcgccgca
ctcctgggta tcctcgccgg ggcgttcgcg 60aacagtgcga tcgaccgcgt gcgcctggag
accgcgtgcg ccgagccgaa gtcgaccccc 120accggctcaa ccccgccgcc
cccctcccct gcgtccgcgg tagccacccg gatcgcgatg 180atcgacacca
tcacgcgacg acacgacatc agtgcccgcc gcgtgctcgt cgaactcgca
240acggccctcc tgttcgtcgg gatcactctc cgtctcgccg ctctcggtct
tctcccggca 300acaccggcct atctcttgca aacggctgcc gaacttcctc
gtcgtaccgt cgtacccgat 360cgtattcgcc tgcctttcag tgggttccgt
cgtgccgttc tgttcggggt ctacttcgta 420ctagccctga tctatccggc
cggcatgggg ttcggcgacg tcaaacttgc cggcgtcatc 480ggcgccgtcc
tcgcctacct gtcgtacggc acattgctcg tcggggcgtt tctcgcgttc
540ctggtggccg cactcgtcgg cctgatcatc ctggtcaccc gtcgcggtcg
gatcgggacc 600acgattccct tcgggccgta catgattgcg gcggccatcg
ttgcgatcct ggcggccgat 660ccgctggcgc gcgcgtatct ggactgggcc
gccgcggcct ga 7022207DNARhodococcus equi 2atgaacctct tcttcgcgaa
cctgtacctc atgggcttag acgtcaagga ccgtctgacc 60cgtgacgacc gcggcgccac
tgcggtcgag tacggactga tggtcgccgg catcgcgatg 120gtgatcattg
ttgcggtttt cgccttcggc gataagatta ccgacctctt cgatggcttc
180aacttcgacg atcccggcgg cgagtag 2073402DNARhodococcus equi
3atgaagcgcc tcacttccga ttcaggggtc gccgcagtcg aattcgctct cgtcgttccg
60atcctgatca cactggtcct cggcatcgtg gagttcggtc ggggttacaa cgtccagaac
120gcggtcagcg ctgctgcccg cgagggtgca cggacgatgg cgatcaagaa
ggatccggcg 180gcggcgcgtg ctgccgtgaa gggcgcgggt gtgttcagtc
cggcgatcac cgatgcggag 240atctgcatca gcacttcggg aacgcagggc
tgttcggcaa cgtcgtgtcc gagcggaagt 300accgtgacgc tcacggtcag
ctatccactc gagtacatga cgggactctt tcccggtaag 360ccgacgctca
ccggcacggg ggtcatgcga tgcggtgggt ga 4024945DNARhodococcus equi
4atgtcgaatg acgagcgcgg ggtcgtcgcc gtgctcgttg cgatcctcat ggtcgtgctc
60ctgggatgtg ctgcgatctc ggtcgacatc ggtgcgaact atgtcgtcaa acgtcagttg
120cagaacgggg ccgatgcggc tgcgctcgcc gtagctcagg aatccagttg
caaggcagga 180tcttccgcct catccgtgtc gagccttgtc caggcgaacg
tcaacagctc gtcggctgca 240agtgcggcgg tgatcgacgg tgtgaagcgg
aaggtgacgg tcactgcgtc ggcggtgggt 300gacgacggcc tcgccggccg
gaggaacgtg ttcgctccgg tcctcggagt cgaccgcagc 360gagatctcgg
cgtctgcgac tgcaagctgc gtgtttcccc tcggggggac cgcggaactc
420ccgctcacgt tccacaagtg ccatttcgac gaatcccgca gtctggacgt
gaagatcctc 480gtcgcctaca acgtgacggc gccgcgctgc aatggaacct
cgggaaatgc ggcaccgggc 540aatttcggct ggctgcaggg ggcgaacggt
cgatgcccgg cgaagatcga cgccgccgtc 600tacgcaacac cgggcgacac
cggtaacaac attccggggc cgtgcaagga caccatcaag 660cagtttcaga
atgccgtcgt gcgggtcccg atctacgacg tcgcaggtgg aaccggaagc
720ggtggatggt ttcacgtcgt cggtttggct gccttcaaga ttcagggcta
ccggctgagc 780ggcaacccgg agttcaactg gaacaacgat gttcacgggg
cgctgagttg caccggcagc 840tgtcgcggca tcatcggcac cttcgtgaag
attgtcagcc tcgattcgga tctgacgccg 900ggagggatcg atttcggcgt
gagtacgatc agcttgctcg attag 9455738DNARhodococcus equi 5ttgagaaccc
gaatcattgc tgcgatctgt gcgatcgttc tcgcggtcgc gggaaccctc 60gccctgatct
cgtatgtacg cggggccgat gcccgcgccc tggcgggtac acgcaccgtc
120gatgtgctcg tcgccgatca gacgattccg aagaacactc ccgctgattc
gctcgtggga 180atggttgtgg tcaagaaact tccggaaatg gcggtgctac
ccgatcgggt gaccagtctc 240gaccaactgt ccggcaaggt cgcgctgacc
gacctcctgc ctggcgaaca actggtctcg 300gcgcgattcg tcgacccggc
gaccgcccga agtcaggacc agggaggaat ccccgagggg 360atgcaggagg
tgacggttct tctcgagccg caacgcgcac tgggaggcca catcgcgtcg
420ggcgataccg tcggcgtctt catgtccttc tcgccgcccg tcaagaacta
cgaaacacat 480ctgagattgc agaaagtgcg agtcacgcgg gtccagggaa
cgttctccaa cgccgacgaa 540ggggattcgg ccacggtcga ctcgtcgccg
agccctgctc ccaccgaggc ctttctcgtc 600tcgctggcgg tcgacgtgcc
gatggcggag cgcgtcgttt tcgccgcgga gcacgggacc 660atctggcttt
ccaatgagcc gccgagttcg aacgaggccg gggcatccgt ggtctccccg
720gaaggagtgt tccgatga 73861200DNARhodococcus equi 6atgagccgca
tcgtcctgct gaccgatcgc gacgatttcg cccgccgcgt gtaccacgcc 60gcggacggca
accttctggt gttgccggcg cagccggttc cccgggggcc ggcgcagttg
120gtcgggctcg gcgtgaccgt gcaaccagaa gttctcgttc tcggtccgga
cgtgccggaa 180gtggagggcc tctccctcgc cggccggatc gatcattcga
cgcccggcac cacggtggtt 240ctggccagtg atgcgggcac cgacgtgtgg
ttgcgggcga tgcgcgccgg cgtgcgggac 300gtgatgtcgc cggaggcgga
gatcgcggac gttcgtgcgg tactcgatcg agcgggccag 360gccgcactgg
cgcgacgtca gggggcgagt gcaccggcgg agcagcatgc ggttcaaggg
420aaggtcatcg tggtcgcgtc gccgaaaggc ggaaccggaa agaccaccgt
tgcgacgaat 480cttgcagtag gactcgcggc ggcagcgcct cactcgacgg
tgttggtgga cctcgacgtg 540cagttcgggg acgttgccag tgctctccag
ttggttccgg aacattgcct gaccgacgcc 600gtcgcgggcc cggccagcca
ggacatgatc gtcctcaaga ccgtcctgac accccattcc 660acaggactgc
atgcgctgtg tgggtcggac tcgcccgcgg cgggcgacag catcaccggc
720gagcaggtga gcactctgct gacgcagttg gcggccgaat tccggtacgt
ggtcgtcgac 780accgcgcccg gtttgctcga acacaccctg gcggcgctcg
accttgctac cgacgtcgtg 840ttggtgtcgg gtatggacgt gcccagcgtc
cgcgggatgc acaaggaact gcaattgctg 900acggagctga atctgggtcc
ggtcgtgcgg catgtcgtgc tcaactttgc ggatcgacgc 960gaggggctga
cggtccagga catccagaac accatcgggg tccccgccga tatcgtgatc
1020aagcgctcga aagccgttgc cctctcgacg aaccgggggg ttccactgct
tcagaacccg 1080ggtcgggatc gcactgcgaa agagttgtgg cgactcgtcg
gccgtatcga tccggctccc 1140gataccgcca agggtggacg cgcgcggcat
cgggcagccg aggcggtggg tgcgaaatga 120071398DNARhodococcus equi
7atgagactgt cccaacggct cgaggccgtg cgcggagccg cacccgtcga agccgccgca
60ccgatcccgc cggggaagca ggggaaggcg aaaacgtccc tccctccggc cgacgctctc
120gccgaactga aggaccgtgc gagtgcggcc ctgtacaccc ggatcggcac
ccgcttcaac 180gactcctcgt tgagcgagga gcaactgcat ctcctggtcc
gtgaggaact ggccgaaatc 240gtggagcagg agacgacgcc actcaccttc
gacgaacggc agcgcctgct ccgtgaggtt 300gccgacgagg tactggggca
cggaccgctc cagcggctac tggaggaccc gtcggtcacc 360gagatcatgg
tcaacagcca cgacatggtc tacgtcgagc gggacggcac cctcgtccgc
420agctccgcgc gattcgcgga cgaggcgcac ctgcgtcgcg tgatcgaacg
catcgtttcc 480gccgtcggtc gacggatcga cgaatcgtcc ccgctcgtgg
atgcacgctt ggcggatggc 540tcccgtgtca acgcggtgat cccaccgctc
gcattcaacg gctcctcgct caccattcga 600aagttctcga aagatccgtt
ccaggtcgac gatctcatcg ccttcggcac tctctcgcac 660gagatggccg
aactgctcga cgcgtgtgtg caggcgcgac tgaacgtcat cgtctcgggc
720ggcacgggca cggggaagac gacgctgctc aacgtgctct cgtcgttcat
tccggaaggg 780gagcggatcg tcaccatcga ggacgccgtg gaactgcaac
ttcagcagga ccacgtcgta 840cggttggaga gccgaccgcc gaacatcgag
ggcaagggtg ccgtcaccat ccgcgacctg 900gtgcggaact cgctgcgtat
gcgtcccgac cgcatcgtgg tgggggagtg tcgcggaggc 960gagagtctcg
acatgctgca agcgatgaac accggtcacg acgggtcgct gtcgacggtg
1020catgcgaatt cgccccgtga cgccatcgcg cgcttggaga cgctcgtgtt
gatggccggc 1080atggacctgc cgttgcgggc gatccgggag cagattgctt
cggcggtcga cgtgatcgtg 1140cagctcactc gactacgtga cggcactcgg
cgagtgaccc acgtgaccga ggtccagggc 1200atggagggtg agatcgtcac
cctgcaggat gccttcctgt tcgactacag cgccggcgtc 1260gacgcgcgcg
ggcgattcct cggcagaccg cagccgaccg gagtgcggcc gcggttcacc
1320gacagattcc gagatctcgg tattgctttg tcgccgagtg ttttcggggt
gggagaaccc 1380tcccgggggc gggtatga 139881869DNARhodococcus equi
8atgagccggt gcgtggtggc cgtcgtgctc gccctcggtg cgggtgttct gggaattccc
60gccgtagccg cggcggccga ggaggctgtc caggtctcgg cggtcgacac gacccggttt
120cccgacatcg aggtgtccat cctcgcgccg cccggtatcg aagggcaggc
gatcgatccg 180ggaacgttcg cgctcaccga gggcggcgtg ccgcgagaga
tcgaggtcag gcagcagccg 240ggttccgagc aggacatcgt gctcgcaatc
gacgtgtccg ggggcatgtc gggtccggcg 300ctggacgacg tgaagcgcgc
cgcatcggat ttcgtgcggc aggcgccggc cggcgcccac 360atcggaatcg
tcgcgatctc gtcgacgcca caggtgctct cggaactgac gacggactcc
420gaggacctgc tccgcaggat cgacggactg aaggcgggcg gcaacagcgc
gatcgcagat 480tcggtggtga ccgccgccga gatgctcgag cgcggcgaag
cggccaacaa catcctgctt 540ctgttgacgg acggcgccga cacgtcgagt
gcacactcga tgtcggaact cccgtccgtc 600ctgagtcggt cgcgcgcgtc
gctgtacgcc gtgcagatgt cgacacccga gacgaactct 660gctctcctgc
agcaggttgc gcgggagtcg cgcggtcagt acgcgtctgc gggtgatacg
720gcggcgctgg gtgcgatcta ccagtcggcc gctcgcgcgc tcggaaacct
gtacgtcgtc 780cgataccgat cggaagcgaa tggcgatacc caggtggtgg
cgagcgtgcg cagcggcgca 840gccggccgag tgagcgatcc gttcccggtg
acattgcccg gtgtggtgcc gacgccgagc 900gtcgtcgccg ggaccgtcga
cggtttcttc acgtcttcga cggggctggt gatcgggctc 960ctagcgtgct
actcggcgct tgcgggaggc gtgctggcgg tcgccggtag agcgcccgcg
1020aggatttcgg cagcacgtcg tgggcggcag gacggacggg actcgatgct
gtcccgattc 1080gcggaacggc tggtgcagtg gatcgatcag aacctgagga
gacgcggacg catcgctgcc 1140cgcacccagg cgctacagga ggcggggctg
aagcttcgtc caggtgactt catcgccctg 1200gtcggtgctg cggcgatcac
cgctgcggcg atcggtctcc tggcttcggg catcgtggcg 1260gcgctcttgc
tcgcggcgat cacagtggga ttgtcgagaa tctatctccg tgtgatggcc
1320ggtaggcgtc gggccgcgtt cgctgatcag ctcgacgatt ccctgcagct
gctggccagc 1380aatctccgag ccgggcacag catgctccga gcgctcgatt
ccctttcccg agaggcggag 1440gtgccgactt cggaggagtt cgctcggatc
gtcaacgaga ctcgggtggg acgtgatctc 1500aacgagtctc tcgacgacgt
ggcccggcgg atgcgaagtg acgatttcaa ctggatagct 1560caggcaatcg
ccatcaaccg tgaggtcgga ggcgacctcg cggaagtcct cgaccaggtg
1620ggcaacacca ttcgagagcg aaatcagatt cgacggcagg tgaaagccct
tgctgccgag 1680gggaaactgt ccgcctacgt gctgatggcg ctgcccttcg
gtctcaccgc atttctgctc 1740gtctcgaatc cggactacct gtcgaagttg
acgggtagcg ccatcggcta cgtgatgatc 1800gcggtggggc tcgtcatgct
gaccgtcggt gggctgtgga tgaacaaggt tgtctcggtc 1860aagttctag
18699891DNARhodococcus equi 9gtgattccac cgctggtgct catggcggcg
ctgtccgtcg gcggggcgtt gggtgttctg 60gtgtggttga cggtcggcgc ccgagatccg
gaacgcggac ccgcccttcg gaacctgcag 120tcgcagctgg cgttgccgat
tccggagtcg ggaggcgcgc caccgctttc gctcggccga 180ttcgtgaagc
tgctgtcgcc gcccgggacg atggcccgct tggaacgact gcacatcctt
240gccggtcgtc cagcggcgtg ggttccggaa cgggccgcga tggcgaagat
cgttctcgcc 300gcggccgccg ccctgctcgg ccttctcgcg gtgggtgcgt
cgcctggcgt cggccgggtg 360ctgttcgctg cggccgccgt cgcgctggcg
tatttcgtcc cggaacttct cctgcagagc 420agggggcagg agcgccaagc
cgcgatcgaa ctggcgcttg ccgacaccct cgaccagatg 480acgatcgcag
tcgaggcggg cctggggttc gaagccgcca tgcagcgggc cgcgaagaac
540ggaaaggggc cgctggccga ggaattcatc cggacattgc aggacataca
gatggggcag 600tcgaggcgaa tcgcgtacct ggatcttgcc gccagaacga
aagcacccaa cttgcggagg 660ttccttcggg ccgtcatcca agccgacgag
tacggcgtgg ccatcgccga ggtcctgcgg 720acccaggcct cggagatgcg
tctgaaacgc cgtcagagtg ctgaggagaa ggcgatgaag 780gttccggtga
aggtgctgtt tccgttgatg acctgcatcc tgccgaccat cttcatcgtg
840atcctgggtc cggcggtgat caacatgatg gaggtcttgg gcggtatgta a
8911068PRTRhodococcus equi 10Met Asn Leu Phe Phe Ala Asn Leu Tyr
Leu Met Gly Leu Asp Val Lys1 5 10 15Asp Arg Leu Thr Arg Asp Asp Arg
Gly Ala Thr Ala Val Glu Tyr Gly 20 25 30Leu Met Val Ala Gly Ile Ala
Met Val Ile Ile Val Ala Val Phe Ala 35 40 45Phe Gly Asp Lys Ile Thr
Asp Leu Phe Asp Gly Phe Asn Phe Asp Asp 50 55 60Pro Gly Gly
Glu651118PRTRhodococcus equi 11Asp Lys Ile Thr Asp Leu Phe Asp Gly
Phe Asn Phe Asp Asp Pro Gly1 5 10 15Gly Glu12262PRTRhodococcus equi
12Val Ile Val Ala Ala Gly Val Gly Ala Ala Leu Leu Gly Ile Leu Ala1
5 10 15Gly Ala Phe Ala Asn Ser Ala Ile Asp Arg Val Arg Leu Glu Thr
Ala 20 25 30Cys Ala Glu Pro Lys Ser Thr Pro Thr Gly Ser Thr Pro Pro
Pro Pro 35 40 45Ser Pro Ala Ser Ala Val Ala Thr Arg Ile Ala Met Ile
Asp Thr Ile 50 55 60Thr Arg Arg His Asp Ile Ser Ala Arg Arg Val Leu
Val Glu Leu Ala65 70 75 80Thr Ala Leu Leu Phe Val Gly Ile Thr Leu
Arg Leu Ala Ala Leu Gly 85 90 95Leu Leu Pro Ala Thr Pro Ala Tyr Leu
Trp Phe Ala Ala Val Gly Ile 100 105 110Ala Leu Ala Val Ile Asp Ile
Asp Cys Lys Arg Leu Pro Asn Phe Leu 115 120 125Val Val Pro Ser Tyr
Pro Ile Val Phe Ala Cys Leu Ser Val Gly Ser 130 135 140Val Val Thr
Gly Asp Trp Ser Ala Leu Leu Arg Ala Ala Ile Gly Ala145 150 155
160Ala Val Leu Phe Gly Val Tyr Phe Val Leu Ala Leu Ile Tyr Pro Ala
165 170 175Gly Met Gly Phe Gly Asp Val Lys Leu Ala Gly Val Ile Gly
Ala Val 180 185 190Leu Ala Tyr Leu Ser Tyr Gly Thr Leu Leu Val Gly
Ala Phe Leu Ala 195 200 205Phe Leu Val Ala Ala Leu Val Gly Leu Ile
Ile Leu Val Thr Arg Arg 210 215 220Gly Arg Ile Gly Thr Thr Ile Pro
Phe Gly Pro Tyr Met Ile Ala Ala225 230 235 240Ala Ile Val Ala Ile
Leu Ala Ala Asp Pro Leu Ala Arg Ala Tyr Leu 245 250 255Asp Trp Ala
Ala Ala Ala 2601368PRTRhodococcus equi 13Met Asn Leu Phe Phe Ala
Asn Leu Tyr Leu Met Gly Leu Asp Val Lys1 5 10 15Asp Arg Leu Thr Arg
Asp Asp Arg Gly Ala Thr Ala Val Glu Tyr Gly 20 25 30Leu Met Val Ala
Gly Ile Ala Met Val Ile Ile Val Ala Val Phe Ala 35 40 45Phe Gly Asp
Lys Ile Thr Asp Leu Phe Asp Gly Phe Asn Phe Asp Asp 50 55 60Pro Gly
Gly Glu6514133PRTRhodococcus equi 14Met Lys Arg Leu Thr Ser Asp Ser
Gly Val Ala Ala Val Glu Phe Ala1 5 10 15Leu Val Val Pro Ile Leu Ile
Thr Leu Val Leu Gly Ile Val Glu Phe 20 25 30Gly Arg Gly Tyr Asn Val
Gln Asn Ala Val Ser Ala Ala Ala Arg Glu 35 40 45Gly Ala Arg Thr Met
Ala Ile Lys Lys Asp Pro Ala Ala Ala Arg Ala 50 55 60Ala Val Lys Gly
Ala Gly Val Phe Ser Pro Ala Ile Thr Asp Ala Glu65 70 75 80Ile Cys
Ile Ser Thr Ser Gly Thr Gln Gly Cys Ser Ala Thr Ser Cys 85 90 95Pro
Ser Gly Ser Thr Val Thr Leu Thr Val Ser Tyr Pro Leu Glu Tyr 100 105
110Met Thr Gly Leu Phe Pro Gly Lys Pro Thr Leu Thr Gly Thr Gly Val
115 120 125Met Arg Cys Gly Gly 13015314PRTRhodococcus equi 15Met
Ser Asn Asp Glu Arg Gly Val Val Ala Val Leu Val Ala Ile Leu1 5 10
15Met Val Val Leu Leu Gly Cys Ala Ala Ile Ser Val Asp Ile Gly Ala
20 25 30Asn Tyr Val Val Lys Arg Gln Leu Gln Asn Gly Ala Asp Ala Ala
Ala 35 40 45Leu Ala Val Ala Gln Glu Ser Ser Cys Lys Ala Gly Ser Ser
Ala Ser 50 55 60Ser Val Ser Ser Leu Val Gln Ala Asn Val Asn Ser Ser
Ser Ala Ala65 70 75 80Ser Ala Ala Val Ile Asp Gly Val Lys Arg Lys
Val Thr Val Thr Ala 85 90 95Ser Ala Val Gly Asp Asp Gly Leu Ala Gly
Arg Arg Asn Val Phe Ala 100 105 110Pro Val Leu Gly Val Asp Arg Ser
Glu Ile Ser Ala Ser Ala Thr Ala 115 120 125Ser Cys Val Phe Pro Leu
Gly Gly Thr Ala Glu Leu Pro Leu Thr Phe 130 135 140His Lys Cys His
Phe Asp Glu Ser Arg Ser Leu Asp Val Lys Ile Leu145 150 155 160Val
Ala Tyr Asn Val Thr Ala Pro Arg Cys Asn Gly Thr Ser Gly Asn 165 170
175Ala Ala Pro Gly Asn Phe Gly Trp Leu Gln Gly Ala Asn Gly Arg Cys
180 185 190Pro Ala Lys Ile Asp Ala Ala Val Tyr Ala Thr Pro Gly Asp
Thr Gly 195 200 205Asn Asn Ile Pro Gly Pro Cys Lys Asp Thr Ile Lys
Gln Phe Gln Asn 210 215 220Ala Val Val Arg Val Pro Ile Tyr Asp Val
Ala Gly Gly Thr Gly Ser225 230 235 240Gly Gly Trp Phe His Val Val
Gly Leu Ala Ala Phe Lys Ile Gln Gly 245 250 255Tyr Arg Leu Ser Gly
Asn Pro Glu Phe Asn Trp Asn Asn Asp Val His 260 265 270Gly Ala Leu
Ser Cys Thr Gly Ser Cys Arg Gly Ile Ile Gly Thr Phe 275 280 285Val
Lys Ile Val Ser Leu Asp Ser Asp Leu Thr Pro Gly Gly Ile Asp 290 295
300Phe Gly Val Ser Thr Ile Ser Leu Leu Asp305
31016245PRTRhodococcus equi 16Leu Arg Thr Arg Ile Ile Ala Ala Ile
Cys Ala Ile Val Leu Ala Val1 5 10 15Ala Gly Thr Leu Ala Leu Ile Ser
Tyr Val Arg Gly Ala Asp Ala Arg 20 25 30Ala Leu Ala Gly Thr Arg Thr
Val Asp Val Leu Val Ala Asp Gln Thr 35 40 45Ile Pro Lys Asn Thr Pro
Ala Asp Ser Leu Val Gly Met Val Val Val 50 55 60Lys Lys Leu Pro Glu
Met Ala Val Leu Pro Asp Arg Val Thr Ser Leu65 70 75 80Asp Gln Leu
Ser Gly Lys Val Ala Leu Thr Asp Leu Leu Pro Gly Glu 85 90 95Gln Leu
Val Ser Ala Arg Phe Val Asp Pro Ala Thr Ala Arg Ser Gln 100 105
110Asp Gln Gly Gly Ile Pro Glu Gly Met Gln Glu Val Thr Val Leu Leu
115 120 125Glu Pro Gln Arg Ala Leu Gly Gly His Ile Ala Ser Gly Asp
Thr Val 130 135 140Gly Val Phe Met Ser Phe Ser Pro Pro Val Lys Asn
Tyr Glu Thr His145 150
155 160Leu Arg Leu Gln Lys Val Arg Val Thr Arg Val Gln Gly Thr Phe
Ser 165 170 175Asn Ala Asp Glu Gly Asp Ser Ala Thr Val Asp Ser Ser
Pro Ser Pro 180 185 190Ala Pro Thr Glu Ala Phe Leu Val Ser Leu Ala
Val Asp Val Pro Met 195 200 205Ala Glu Arg Val Val Phe Ala Ala Glu
His Gly Thr Ile Trp Leu Ser 210 215 220Asn Glu Pro Pro Ser Ser Asn
Glu Ala Gly Ala Ser Val Val Ser Pro225 230 235 240Glu Gly Val Phe
Arg 24517399PRTRhodococcus equi 17Met Ser Arg Ile Val Leu Leu Thr
Asp Arg Asp Asp Phe Ala Arg Arg1 5 10 15Val Tyr His Ala Ala Asp Gly
Asn Leu Leu Val Leu Pro Ala Gln Pro 20 25 30Val Pro Arg Gly Pro Ala
Gln Leu Val Gly Leu Gly Val Thr Val Gln 35 40 45Pro Glu Val Leu Val
Leu Gly Pro Asp Val Pro Glu Val Glu Gly Leu 50 55 60Ser Leu Ala Gly
Arg Ile Asp His Ser Thr Pro Gly Thr Thr Val Val65 70 75 80Leu Ala
Ser Asp Ala Gly Thr Asp Val Trp Leu Arg Ala Met Arg Ala 85 90 95Gly
Val Arg Asp Val Met Ser Pro Glu Ala Glu Ile Ala Asp Val Arg 100 105
110Ala Val Leu Asp Arg Ala Gly Gln Ala Ala Leu Ala Arg Arg Gln Gly
115 120 125Ala Ser Ala Pro Ala Glu Gln His Ala Val Gln Gly Lys Val
Ile Val 130 135 140Val Ala Ser Pro Lys Gly Gly Thr Gly Lys Thr Thr
Val Ala Thr Asn145 150 155 160Leu Ala Val Gly Leu Ala Ala Ala Ala
Pro His Ser Thr Val Leu Val 165 170 175Asp Leu Asp Val Gln Phe Gly
Asp Val Ala Ser Ala Leu Gln Leu Val 180 185 190Pro Glu His Cys Leu
Thr Asp Ala Val Ala Gly Pro Ala Ser Gln Asp 195 200 205Met Ile Val
Leu Lys Thr Val Leu Thr Pro His Ser Thr Gly Leu His 210 215 220Ala
Leu Cys Gly Ser Asp Ser Pro Ala Ala Gly Asp Ser Ile Thr Gly225 230
235 240Glu Gln Val Ser Thr Leu Leu Thr Gln Leu Ala Ala Glu Phe Arg
Tyr 245 250 255Val Val Val Asp Thr Ala Pro Gly Leu Leu Glu His Thr
Leu Ala Ala 260 265 270Leu Asp Leu Ala Thr Asp Val Val Leu Val Ser
Gly Met Asp Val Pro 275 280 285Ser Val Arg Gly Met His Lys Glu Leu
Gln Leu Leu Thr Glu Leu Asn 290 295 300Leu Gly Pro Val Val Arg His
Val Val Leu Asn Phe Ala Asp Arg Arg305 310 315 320Glu Gly Leu Thr
Val Gln Asp Ile Gln Asn Thr Ile Gly Val Pro Ala 325 330 335Asp Ile
Val Ile Lys Arg Ser Lys Ala Val Ala Leu Ser Thr Asn Arg 340 345
350Gly Val Pro Leu Leu Gln Asn Pro Gly Arg Asp Arg Thr Ala Lys Glu
355 360 365Leu Trp Arg Leu Val Gly Arg Ile Asp Pro Ala Pro Asp Thr
Ala Lys 370 375 380Gly Gly Arg Ala Arg His Arg Ala Ala Glu Ala Val
Gly Ala Lys385 390 39518465PRTRhodococcus equi 18Met Arg Leu Ser
Gln Arg Leu Glu Ala Val Arg Gly Ala Ala Pro Val1 5 10 15Glu Ala Ala
Ala Pro Ile Pro Pro Gly Lys Gln Gly Lys Ala Lys Thr 20 25 30Ser Leu
Pro Pro Ala Asp Ala Leu Ala Glu Leu Lys Asp Arg Ala Ser 35 40 45Ala
Ala Leu Tyr Thr Arg Ile Gly Thr Arg Phe Asn Asp Ser Ser Leu 50 55
60Ser Glu Glu Gln Leu His Leu Leu Val Arg Glu Glu Leu Ala Glu Ile65
70 75 80Val Glu Gln Glu Thr Thr Pro Leu Thr Phe Asp Glu Arg Gln Arg
Leu 85 90 95Leu Arg Glu Val Ala Asp Glu Val Leu Gly His Gly Pro Leu
Gln Arg 100 105 110Leu Leu Glu Asp Pro Ser Val Thr Glu Ile Met Val
Asn Ser His Asp 115 120 125Met Val Tyr Val Glu Arg Asp Gly Thr Leu
Val Arg Ser Ser Ala Arg 130 135 140Phe Ala Asp Glu Ala His Leu Arg
Arg Val Ile Glu Arg Ile Val Ser145 150 155 160Ala Val Gly Arg Arg
Ile Asp Glu Ser Ser Pro Leu Val Asp Ala Arg 165 170 175Leu Ala Asp
Gly Ser Arg Val Asn Ala Val Ile Pro Pro Leu Ala Phe 180 185 190Asn
Gly Ser Ser Leu Thr Ile Arg Lys Phe Ser Lys Asp Pro Phe Gln 195 200
205Val Asp Asp Leu Ile Ala Phe Gly Thr Leu Ser His Glu Met Ala Glu
210 215 220Leu Leu Asp Ala Cys Val Gln Ala Arg Leu Asn Val Ile Val
Ser Gly225 230 235 240Gly Thr Gly Thr Gly Lys Thr Thr Leu Leu Asn
Val Leu Ser Ser Phe 245 250 255Ile Pro Glu Gly Glu Arg Ile Val Thr
Ile Glu Asp Ala Val Glu Leu 260 265 270Gln Leu Gln Gln Asp His Val
Val Arg Leu Glu Ser Arg Pro Pro Asn 275 280 285Ile Glu Gly Lys Gly
Ala Val Thr Ile Arg Asp Leu Val Arg Asn Ser 290 295 300Leu Arg Met
Arg Pro Asp Arg Ile Val Val Gly Glu Cys Arg Gly Gly305 310 315
320Glu Ser Leu Asp Met Leu Gln Ala Met Asn Thr Gly His Asp Gly Ser
325 330 335Leu Ser Thr Val His Ala Asn Ser Pro Arg Asp Ala Ile Ala
Arg Leu 340 345 350Glu Thr Leu Val Leu Met Ala Gly Met Asp Leu Pro
Leu Arg Ala Ile 355 360 365Arg Glu Gln Ile Ala Ser Ala Val Asp Val
Ile Val Gln Leu Thr Arg 370 375 380Leu Arg Asp Gly Thr Arg Arg Val
Thr His Val Thr Glu Val Gln Gly385 390 395 400Met Glu Gly Glu Ile
Val Thr Leu Gln Asp Ala Phe Leu Phe Asp Tyr 405 410 415Ser Ala Gly
Val Asp Ala Arg Gly Arg Phe Leu Gly Arg Pro Gln Pro 420 425 430Thr
Gly Val Arg Pro Arg Phe Thr Asp Arg Phe Arg Asp Leu Gly Ile 435 440
445Ala Leu Ser Pro Ser Val Phe Gly Val Gly Glu Pro Ser Arg Gly Arg
450 455 460Val46519622PRTRhodococcus equi 19Met Ser Arg Cys Val Val
Ala Val Val Leu Ala Leu Gly Ala Gly Val1 5 10 15Leu Gly Ile Pro Ala
Val Ala Ala Ala Ala Glu Glu Ala Val Gln Val 20 25 30Ser Ala Val Asp
Thr Thr Arg Phe Pro Asp Ile Glu Val Ser Ile Leu 35 40 45Ala Pro Pro
Gly Ile Glu Gly Gln Ala Ile Asp Pro Gly Thr Phe Ala 50 55 60Leu Thr
Glu Gly Gly Val Pro Arg Glu Ile Glu Val Arg Gln Gln Pro65 70 75
80Gly Ser Glu Gln Asp Ile Val Leu Ala Ile Asp Val Ser Gly Gly Met
85 90 95Ser Gly Pro Ala Leu Asp Asp Val Lys Arg Ala Ala Ser Asp Phe
Val 100 105 110Arg Gln Ala Pro Ala Gly Ala His Ile Gly Ile Val Ala
Ile Ser Ser 115 120 125Thr Pro Gln Val Leu Ser Glu Leu Thr Thr Asp
Ser Glu Asp Leu Leu 130 135 140Arg Arg Ile Asp Gly Leu Lys Ala Gly
Gly Asn Ser Ala Ile Ala Asp145 150 155 160Ser Val Val Thr Ala Ala
Glu Met Leu Glu Arg Gly Glu Ala Ala Asn 165 170 175Asn Ile Leu Leu
Leu Leu Thr Asp Gly Ala Asp Thr Ser Ser Ala His 180 185 190Ser Met
Ser Glu Leu Pro Ser Val Leu Ser Arg Ser Arg Ala Ser Leu 195 200
205Tyr Ala Val Gln Met Ser Thr Pro Glu Thr Asn Ser Ala Leu Leu Gln
210 215 220Gln Val Ala Arg Glu Ser Arg Gly Gln Tyr Ala Ser Ala Gly
Asp Thr225 230 235 240Ala Ala Leu Gly Ala Ile Tyr Gln Ser Ala Ala
Arg Ala Leu Gly Asn 245 250 255Leu Tyr Val Val Arg Tyr Arg Ser Glu
Ala Asn Gly Asp Thr Gln Val 260 265 270Val Ala Ser Val Arg Ser Gly
Ala Ala Gly Arg Val Ser Asp Pro Phe 275 280 285Pro Val Thr Leu Pro
Gly Val Val Pro Thr Pro Ser Val Val Ala Gly 290 295 300Thr Val Asp
Gly Phe Phe Thr Ser Ser Thr Gly Leu Val Ile Gly Leu305 310 315
320Leu Ala Cys Tyr Ser Ala Leu Ala Gly Gly Val Leu Ala Val Ala Gly
325 330 335Arg Ala Pro Ala Arg Ile Ser Ala Ala Arg Arg Gly Arg Gln
Asp Gly 340 345 350Arg Asp Ser Met Leu Ser Arg Phe Ala Glu Arg Leu
Val Gln Trp Ile 355 360 365Asp Gln Asn Leu Arg Arg Arg Gly Arg Ile
Ala Ala Arg Thr Gln Ala 370 375 380Leu Gln Glu Ala Gly Leu Lys Leu
Arg Pro Gly Asp Phe Ile Ala Leu385 390 395 400Val Gly Ala Ala Ala
Ile Thr Ala Ala Ala Ile Gly Leu Leu Ala Ser 405 410 415Gly Ile Val
Ala Ala Leu Leu Leu Ala Ala Ile Thr Val Gly Leu Ser 420 425 430Arg
Ile Tyr Leu Arg Val Met Ala Gly Arg Arg Arg Ala Ala Phe Ala 435 440
445Asp Gln Leu Asp Asp Ser Leu Gln Leu Leu Ala Ser Asn Leu Arg Ala
450 455 460Gly His Ser Met Leu Arg Ala Leu Asp Ser Leu Ser Arg Glu
Ala Glu465 470 475 480Val Pro Thr Ser Glu Glu Phe Ala Arg Ile Val
Asn Glu Thr Arg Val 485 490 495Gly Arg Asp Leu Asn Glu Ser Leu Asp
Asp Val Ala Arg Arg Met Arg 500 505 510Ser Asp Asp Phe Asn Trp Ile
Ala Gln Ala Ile Ala Ile Asn Arg Glu 515 520 525Val Gly Gly Asp Leu
Ala Glu Val Leu Asp Gln Val Gly Asn Thr Ile 530 535 540Arg Glu Arg
Asn Gln Ile Arg Arg Gln Val Lys Ala Leu Ala Ala Glu545 550 555
560Gly Lys Leu Ser Ala Tyr Val Leu Met Ala Leu Pro Phe Gly Leu Thr
565 570 575Ala Phe Leu Leu Val Ser Asn Pro Asp Tyr Leu Ser Lys Leu
Thr Gly 580 585 590Ser Ala Ile Gly Tyr Val Met Ile Ala Val Gly Leu
Val Met Leu Thr 595 600 605Val Gly Gly Leu Trp Met Asn Lys Val Val
Ser Val Lys Phe 610 615 62020296PRTRhodococcus equi 20Val Ile Pro
Pro Leu Val Leu Met Ala Ala Leu Ser Val Gly Gly Ala1 5 10 15Leu Gly
Val Leu Val Trp Leu Thr Val Gly Ala Arg Asp Pro Glu Arg 20 25 30Gly
Pro Ala Leu Arg Asn Leu Gln Ser Gln Leu Ala Leu Pro Ile Pro 35 40
45Glu Ser Gly Gly Ala Pro Pro Leu Ser Leu Gly Arg Phe Val Lys Leu
50 55 60Leu Ser Pro Pro Gly Thr Met Ala Arg Leu Glu Arg Leu His Ile
Leu65 70 75 80Ala Gly Arg Pro Ala Ala Trp Val Pro Glu Arg Ala Ala
Met Ala Lys 85 90 95Ile Val Leu Ala Ala Ala Ala Ala Leu Leu Gly Leu
Leu Ala Val Gly 100 105 110Ala Ser Pro Gly Val Gly Arg Val Leu Phe
Ala Ala Ala Ala Val Ala 115 120 125Leu Ala Tyr Phe Val Pro Glu Leu
Leu Leu Gln Ser Arg Gly Gln Glu 130 135 140Arg Gln Ala Ala Ile Glu
Leu Ala Leu Ala Asp Thr Leu Asp Gln Met145 150 155 160Thr Ile Ala
Val Glu Ala Gly Leu Gly Phe Glu Ala Ala Met Gln Arg 165 170 175Ala
Ala Lys Asn Gly Lys Gly Pro Leu Ala Glu Glu Phe Ile Arg Thr 180 185
190Leu Gln Asp Ile Gln Met Gly Gln Ser Arg Arg Ile Ala Tyr Leu Asp
195 200 205Leu Ala Ala Arg Thr Lys Ala Pro Asn Leu Arg Arg Phe Leu
Arg Ala 210 215 220Val Ile Gln Ala Asp Glu Tyr Gly Val Ala Ile Ala
Glu Val Leu Arg225 230 235 240Thr Gln Ala Ser Glu Met Arg Leu Lys
Arg Arg Gln Ser Ala Glu Glu 245 250 255Lys Ala Met Lys Val Pro Val
Lys Val Leu Phe Pro Leu Met Thr Cys 260 265 270Ile Leu Pro Thr Ile
Phe Ile Val Ile Leu Gly Pro Ala Val Ile Asn 275 280 285Met Met Glu
Val Leu Gly Gly Met 290 29521262PRTRhodococcus equi 21Val Ile Val
Ala Ala Gly Val Gly Ala Ala Leu Leu Gly Ile Leu Ala1 5 10 15Gly Ala
Phe Ala Asn Ser Ala Ile Asp Arg Val Arg Leu Glu Thr Ala 20 25 30Cys
Ala Glu Pro Lys Ser Thr Pro Thr Gly Ser Thr Pro Pro Pro Pro 35 40
45Ser Pro Ala Ser Ala Val Ala Thr Arg Ile Ala Met Ile Asp Thr Ile
50 55 60Thr Arg Arg Arg Asp Ile Ser Ala Arg Arg Met Leu Val Glu Leu
Ala65 70 75 80Thr Ala Leu Leu Phe Val Ala Ile Thr Leu Arg Leu Ala
Ala Leu Gly 85 90 95Leu Leu Pro Ala Ala Pro Ala Tyr Leu Trp Phe Ala
Val Ile Gly Ile 100 105 110Ala Leu Ala Val Ile Asp Ile Asp Cys Lys
Arg Leu Pro Asn Phe Leu 115 120 125Val Val Pro Ser Tyr Pro Ile Val
Phe Ala Cys Leu Ala Val Gly Ser 130 135 140Val Val Thr Gly Asp Trp
Ser Ala Leu Leu Arg Ala Ala Ile Gly Ala145 150 155 160Ala Val Leu
Phe Gly Phe Tyr Phe Val Leu Ala Leu Ile Tyr Pro Ala 165 170 175Gly
Met Gly Phe Gly Asp Val Lys Leu Ala Gly Val Ile Gly Ala Val 180 185
190Leu Ala Tyr Leu Ser Tyr Gly Thr Leu Leu Val Gly Ala Phe Leu Ala
195 200 205Phe Leu Val Ala Ala Leu Val Gly Leu Ile Ile Leu Val Thr
Arg Arg 210 215 220Gly Arg Ile Gly Thr Thr Ile Pro Phe Gly Pro Tyr
Met Ile Ala Ala225 230 235 240Ala Val Val Ala Ile Leu Ala Ala Asp
Pro Leu Ala Arg Ala Tyr Leu 245 250 255Asp Trp Ala Ala Ala Ala
26022262PRTRhodococcus equi 22Val Ile Val Ala Ala Gly Val Gly Ala
Ala Leu Leu Gly Ile Leu Ala1 5 10 15Gly Ala Phe Ala Asn Ser Ala Ile
Asp Arg Val Arg Leu Glu Thr Ala 20 25 30Cys Ala Glu Pro Arg Ala Thr
Pro Thr Gly Ser Thr Pro Pro Pro Pro 35 40 45Ser Pro Thr Ser Ala Val
Ala Thr Arg Ile Ala Met Ile Asp Thr Ile 50 55 60Thr Arg Arg Arg Asp
Ile Ser Ala Arg Arg Met Leu Val Glu Leu Ala65 70 75 80Thr Ala Leu
Leu Phe Val Ala Ile Thr Leu Arg Leu Ala Ala Leu Asp 85 90 95Leu Leu
Pro Ala Ala Pro Ala Tyr Leu Trp Phe Ala Val Ile Gly Ile 100 105
110Ala Leu Ala Val Ile Asp Ile Asp Cys Lys Arg Leu Pro Asn Phe Leu
115 120 125Val Val Pro Ser Tyr Pro Ile Val Phe Ala Cys Leu Ala Val
Gly Ser 130 135 140Val Val Thr Gly Asp Trp Ser Ala Leu Leu Arg Ala
Ala Ile Gly Ala145 150 155 160Ala Val Leu Phe Gly Phe Tyr Phe Val
Leu Ala Leu Ile Tyr Pro Ala 165 170 175Gly Met Gly Phe Gly Asp Val
Lys Leu Ala Gly Val Ile Gly Ala Val 180 185 190Leu Ala Tyr Leu Ser
Tyr Gly Thr Leu Leu Val Gly Ala Phe Leu Ala 195 200 205Phe Leu Val
Ala Ala Leu Val Gly Leu Ile Ile Leu Val Thr Arg Arg 210 215 220Gly
Arg Ile Gly Thr Thr Ile Pro Phe Gly Pro Tyr Met Ile Ala Ala225 230
235 240Ala Val Val Ala Ile Leu Ala Ala Asp Pro Leu Ala Arg Ala Tyr
Leu 245 250 255Asp Trp Ala Ala Ala Ala 26023262PRTRhodococcus equi
23Val Ile Val Ala Ala Gly Val Gly Ala Ala Leu Leu Gly Ile Leu Ala1
5 10 15Gly Ala Phe Ala Asn Ser Ala Ile Asp Arg Val Arg Leu Glu Thr
Ala 20 25 30Cys Ala Glu Pro Lys Ser Thr Pro Ala Asn Ser Thr Pro Pro
Ser Pro 35 40 45Ser Pro Thr Ser Ala Val Ala Ala Arg Ile Ala Met Ile
Asp Thr Ile 50 55 60Thr
Arg Arg His Asp Ile Ser Ala Arg Arg Val Leu Val Glu Leu Ala65 70 75
80Thr Ala Leu Leu Phe Val Ala Ile Thr Leu Arg Leu Ala Ala Leu Asp
85 90 95Leu Leu Pro Ala Ala Pro Ala Tyr Leu Trp Phe Ala Val Val Gly
Ile 100 105 110Ala Leu Ala Val Ile Asp Ile Asp Cys Lys Arg Leu Pro
Asn Phe Leu 115 120 125Val Val Pro Ser Tyr Pro Ile Val Phe Ala Cys
Leu Ala Val Gly Ser 130 135 140Val Val Thr Gly Asp Trp Ser Ala Leu
Leu Arg Ala Ala Ile Gly Ala145 150 155 160Ala Val Leu Phe Gly Phe
Tyr Phe Val Leu Ala Leu Ile Tyr Pro Ala 165 170 175Gly Met Gly Phe
Gly Asp Val Lys Leu Ala Gly Val Ile Gly Ala Val 180 185 190Leu Ala
Tyr Leu Ser Tyr Gly Thr Leu Leu Val Gly Ala Phe Leu Ala 195 200
205Phe Leu Val Ala Ala Leu Val Gly Leu Ile Ile Leu Val Thr Arg Arg
210 215 220Gly Arg Ile Gly Thr Thr Ile Pro Phe Gly Pro Tyr Met Ile
Ala Ala225 230 235 240Ala Val Val Ala Ile Leu Ala Ala Asp Pro Leu
Ala Arg Ala Tyr Leu 245 250 255Asp Trp Ala Ala Ala Ala
2602470PRTRhodococcus equi 24Met Asn Leu Phe Phe Ala Asn Leu Tyr
Leu Met Gly Leu Asp Val Lys1 5 10 15Asp Arg Leu Thr Arg Asp Asp Arg
Gly Ala Thr Ala Val Glu Tyr Gly 20 25 30Leu Met Val Ala Gly Ile Ala
Met Val Ile Leu Ile Ala Val Phe Ala 35 40 45Phe Gly Gly Lys Ile Ser
Glu Leu Phe Ser Gly Phe Asn Phe Asp Lys 50 55 60Pro Ala Ala Ser Gly
Thr65 702567PRTRhodococcus equi 25Met Asn Leu Phe Phe Ala Asn Leu
Tyr Leu Met Gly Leu Asp Val Lys1 5 10 15Asp Arg Leu Thr Arg Asp Asp
Arg Gly Ala Thr Ala Val Glu Tyr Gly 20 25 30Leu Met Val Ala Gly Ile
Ala Met Val Ile Ile Ile Ala Val Phe Ala 35 40 45Phe Gly Gly Arg Leu
Ser Thr Leu Phe Gln Asn Phe Asn Phe Ala Asn 50 55 60Pro Gly
Asn652663PRTArtificial SequenceConsensus amino acid sequence of
amino acid sequence alignment of SEQ ID NO 24 and SEQ ID NO 25
26Met Asn Leu Phe Phe Ala Asn Leu Tyr Leu Met Gly Leu Asp Val Lys1
5 10 15Asp Arg Leu Thr Arg Asp Asp Arg Gly Ala Thr Ala Val Glu Tyr
Gly 20 25 30Leu Met Val Ala Gly Ile Ala Met Val Ile Ile Ile Ala Val
Phe Ala 35 40 45Phe Gly Gly Lys Ile Ser Leu Phe Gly Phe Asn Phe Asp
Pro Gly 50 55 6027135PRTRhodococcus equi 27Val Ile Met Lys Arg Leu
Thr Ser Asp Ser Gly Val Ala Ala Val Glu1 5 10 15Phe Ala Leu Val Val
Pro Ile Leu Ile Thr Leu Val Leu Gly Ile Val 20 25 30Glu Phe Gly Arg
Gly Tyr Asn Val Gln Asn Ala Val Ser Ala Ala Ala 35 40 45Arg Glu Gly
Ala Arg Thr Met Ala Ile Lys Lys Asp Pro Ala Ala Ala 50 55 60Arg Ala
Ala Val Lys Gly Ala Gly Val Phe Ser Pro Ala Ile Thr Asp65 70 75
80Ala Glu Ile Cys Ile Ser Thr Ser Gly Ser Gln Gly Cys Ser Ala Thr
85 90 95Ser Cys Pro Ser Gly Ser Thr Val Thr Leu Thr Val Ser Tyr Pro
Leu 100 105 110Glu Tyr Met Thr Gly Leu Phe Pro Gly Lys Pro Thr Leu
Thr Gly Thr 115 120 125Gly Val Met Arg Cys Gly Gly 130
13528130PRTRhodococcus equi 28Leu Arg Ser Asp Ser Gly Val Ala Ala
Val Glu Phe Ala Leu Val Val1 5 10 15Pro Ile Leu Ile Thr Leu Val Leu
Gly Ile Val Glu Phe Gly Arg Gly 20 25 30Tyr Asn Val Gln Asn Ala Val
Ser Ala Ala Ala Arg Glu Gly Ala Arg 35 40 45Thr Met Ala Ile Lys Lys
Asp Pro Ala Ala Ala Arg Ala Ala Val Lys 50 55 60Gly Ala Gly Val Phe
Ser Pro Ala Ile Thr Asp Ala Glu Ile Cys Ile65 70 75 80Ser Thr Ser
Gly Thr Gln Gly Cys Ser Ala Thr Ser Cys Pro Ser Gly 85 90 95Ser Thr
Val Thr Leu Thr Val Ser Tyr Pro Leu Glu Tyr Met Thr Gly 100 105
110Leu Phe Pro Gly Lys Pro Thr Leu Thr Gly Thr Gly Val Met Arg Cys
115 120 125Gly Gly 13029135PRTRhodococcus equi 29Met Gly Met Arg
Arg Phe Gly Ser Asp Ser Gly Ala Ala Ala Val Glu1 5 10 15Phe Ala Leu
Val Val Pro Ile Leu Ile Thr Leu Val Leu Gly Ile Val 20 25 30Glu Phe
Gly Arg Gly Tyr Asn Val Gln Asn Ala Val Ser Ala Ala Ala 35 40 45Arg
Glu Gly Ala Arg Thr Met Ala Ile Lys Lys Asp Pro Ala Ala Ala 50 55
60Arg Ala Ala Val Lys Gly Ala Gly Val Phe Ser Pro Ala Ile Thr Asp65
70 75 80Ala Glu Ile Cys Ile Ser Thr Ser Gly Thr Gln Gly Cys Ser Ala
Thr 85 90 95Ser Cys Pro Ser Gly Ser Thr Val Thr Leu Thr Val Ser Tyr
Pro Leu 100 105 110Glu Tyr Met Thr Gly Leu Phe Pro Gly Lys Pro Thr
Leu Thr Gly Thr 115 120 125Gly Val Met Arg Cys Gly Gly 130
13530321PRTRhodococcus equi 30Met Arg Trp Val Arg Ser Arg Met Ser
Asn Asp Glu Arg Gly Val Val1 5 10 15Ala Val Leu Val Ala Ile Leu Met
Val Val Leu Leu Gly Cys Ala Ala 20 25 30Ile Ser Val Asp Ile Gly Ala
Asn Tyr Val Val Lys Arg Gln Leu Gln 35 40 45Asn Gly Ala Asp Ala Ala
Ala Leu Ala Val Ala Gln Glu Ser Ser Cys 50 55 60Lys Ala Gly Ser Ser
Ala Ser Ser Val Ser Ser Leu Val Gln Ala Asn65 70 75 80Val Asn Ser
Ser Ser Ala Ser Ser Ala Ala Val Ile Asp Gly Val Lys 85 90 95Arg Lys
Val Thr Val Thr Ala Ser Ala Val Gly Asp Asp Gly Leu Ala 100 105
110Gly Arg Arg Asn Val Phe Ala Pro Val Leu Gly Val Asp Arg Ser Glu
115 120 125Ile Ser Ala Ser Ala Thr Ala Ser Cys Val Phe Pro Leu Gly
Gly Thr 130 135 140Ala Glu Leu Pro Leu Thr Phe His Lys Cys His Phe
Asp Glu Ser Arg145 150 155 160Ser Leu Asp Val Lys Ile Leu Val Ala
Tyr Asn Val Thr Ala Pro Arg 165 170 175Cys Asn Gly Thr Ser Gly Asn
Ala Ala Pro Gly Asn Phe Gly Trp Leu 180 185 190Gln Gly Ala Asn Gly
Arg Cys Pro Ala Lys Ile Asp Ala Ala Val Tyr 195 200 205Ala Thr Pro
Gly Asp Thr Gly Asn Asn Ile Pro Gly Pro Cys Lys Asp 210 215 220Thr
Ile Lys Gln Phe Gln Asn Ala Val Val Arg Val Pro Ile Tyr Asp225 230
235 240Val Ala Gly Gly Thr Gly Ser Gly Gly Trp Phe His Val Val Gly
Leu 245 250 255Ala Ala Phe Lys Ile Gln Gly Tyr Arg Leu Ser Gly Asn
Pro Glu Phe 260 265 270Asn Trp Asn Asn Asp Val His Gly Ala Leu Ser
Cys Thr Gly Ser Cys 275 280 285Arg Gly Ile Ile Gly Thr Phe Val Lys
Ile Val Ser Leu Asp Ser Asp 290 295 300Leu Thr Pro Gly Gly Ile Asp
Phe Gly Val Ser Thr Ile Ser Leu Leu305 310 315
320Asp31321PRTRhodococcus equi 31Met Arg Trp Val Arg Ser Arg Met
Ser Asn Asp Glu Arg Gly Val Val1 5 10 15Ala Val Leu Val Ala Ile Leu
Met Val Val Leu Leu Gly Cys Ala Ala 20 25 30Ile Ser Val Asp Ile Gly
Ala Asn Tyr Val Val Lys Arg Gln Leu Gln 35 40 45Asn Gly Ala Asp Ala
Ala Ala Leu Ala Val Ala Gln Glu Ser Asn Cys 50 55 60Lys Ala Gly Ser
Ser Ala Ser Ser Val Ser Ser Leu Val Gln Ala Asn65 70 75 80Val Asn
Ser Ser Ser Ala Ser Ser Ala Ala Val Ile Asp Gly Val Lys 85 90 95Arg
Lys Val Thr Val Thr Ala Ser Ala Val Gly Asp Asp Gly Leu Ala 100 105
110Gly Arg Arg Asn Val Phe Ala Pro Val Leu Gly Val Asp Arg Ser Glu
115 120 125Ile Ser Ala Ser Ala Thr Ala Ser Cys Val Phe Pro Leu Gly
Gly Thr 130 135 140Ala Glu Leu Pro Leu Thr Phe His Lys Cys His Phe
Asp Glu Ser Arg145 150 155 160Ser Leu Asp Val Lys Ile Leu Val Ala
Tyr Asn Val Thr Ala Pro Arg 165 170 175Cys Asn Gly Thr Ser Gly Asn
Ala Ala Pro Gly Asn Phe Gly Trp Leu 180 185 190Gln Gly Ala Asn Gly
Arg Cys Pro Ala Lys Ile Asp Pro Thr Val Tyr 195 200 205Ala Thr Pro
Gly Asp Thr Gly Asn Asn Ile Pro Gly Pro Cys Lys Asp 210 215 220Thr
Ile Lys Gln Phe Gln Asn Ala Val Val Arg Val Pro Ile Tyr Asp225 230
235 240Val Ala Gly Gly Thr Gly Ser Gly Gly Trp Phe His Val Val Gly
Leu 245 250 255Ala Ala Phe Lys Ile Gln Gly Tyr Arg Leu Ser Gly Asn
Pro Glu Phe 260 265 270Asn Trp Asn Asn Asp Val His Gly Ala Leu Ser
Cys Thr Gly Ser Cys 275 280 285Arg Gly Ile Ile Gly Thr Phe Val Lys
Ile Val Ser Leu Asp Ser Asp 290 295 300Leu Thr Pro Gly Gly Ile Asp
Phe Gly Val Ser Thr Ile Ser Leu Leu305 310 315
320Asp32321PRTRhodococcus equi 32Met Arg Trp Val Arg Ser Arg Met
Ser Asn Asp Glu Arg Gly Val Val1 5 10 15Ala Val Phe Val Ala Ile Leu
Met Val Val Leu Leu Gly Cys Ala Ala 20 25 30Ile Ser Val Asp Ile Gly
Ala Asn Tyr Val Val Lys Arg Gln Leu Gln 35 40 45Asn Gly Ala Asp Ala
Ala Ala Leu Ala Val Ala Gln Glu Ser Ser Cys 50 55 60Lys Ala Gly Ser
Ser Ala Ser Ser Val Ser Arg Leu Val Gln Ala Asn65 70 75 80Val Asn
Ser Ser Ser Ala Ser Ser Ala Ala Val Ile Asp Gly Val Lys 85 90 95Arg
Lys Val Thr Val Thr Ala Ser Ala Val Gly Asp Asp Gly Leu Ala 100 105
110Gly Arg Arg Asn Val Phe Ala Pro Val Leu Gly Val Asp Arg Ser Glu
115 120 125Ile Ser Ala Ser Ala Thr Ala Ser Cys Val Phe Pro Leu Gly
Gly Thr 130 135 140Ala Glu Leu Pro Leu Thr Phe His Lys Cys His Phe
Asp Glu Ser Arg145 150 155 160Ser Leu Asp Val Lys Ile Leu Val Ala
Tyr Asn Val Thr Ala Pro Arg 165 170 175Cys Asn Gly Thr Ser Gly Asn
Ala Ala Pro Gly Asn Phe Gly Trp Leu 180 185 190Gln Gly Val Asn Gly
Arg Cys Pro Ala Lys Ile Asp Ala Ala Val Tyr 195 200 205Ala Thr Pro
Gly Asp Thr Gly Asn Asn Ile Pro Gly Pro Cys Lys Asp 210 215 220Thr
Ile Lys Gln Phe Gln Asn Ala Val Val Arg Val Pro Ile Tyr Asp225 230
235 240Val Ala Gly Gly Thr Gly Ser Gly Gly Trp Phe His Val Val Gly
Leu 245 250 255Ala Ala Phe Lys Ile Gln Gly Tyr Arg Leu Ser Gly Asn
Pro Glu Phe 260 265 270Asn Trp Asn Asn Asp Val His Gly Ala Leu Ser
Cys Thr Gly Ser Cys 275 280 285Arg Gly Ile Ile Gly Thr Phe Val Lys
Ile Val Ser Leu Asp Ser Asp 290 295 300Leu Thr Pro Gly Gly Ile Asp
Phe Gly Val Ser Thr Ile Ser Leu Leu305 310 315
320Asp33245PRTRhodococcus equi 33Leu Arg Thr Arg Ile Ile Ala Ala
Ile Cys Ala Ile Val Leu Ala Val1 5 10 15Ala Gly Thr Leu Ala Leu Ile
Ser Tyr Val Arg Gly Ala Asp Ala Arg 20 25 30Ala Leu Ala Gly Thr Arg
Thr Val Asp Val Leu Val Ala Asp Gln Thr 35 40 45Ile Pro Lys Asn Thr
Pro Ala Asp Ser Leu Val Gly Met Val Val Val 50 55 60Lys Lys Leu Pro
Glu Met Ala Val Leu Pro Glu Arg Val Thr Ser Leu65 70 75 80Asp Gln
Leu Ser Gly Lys Val Ala Leu Thr Asp Leu Leu Pro Gly Glu 85 90 95Gln
Leu Val Ser Ala Arg Phe Ala Asp Pro Ala Thr Ala Arg Ser Gln 100 105
110Asp Gln Gly Gly Ile Pro Glu Gly Met Gln Glu Val Thr Val Leu Leu
115 120 125Glu Pro Gln Arg Ala Leu Gly Gly His Ile Ala Ser Gly Asp
Thr Val 130 135 140Gly Val Phe Met Ser Phe Ser Pro Pro Val Lys Asn
Tyr Glu Thr His145 150 155 160Leu Arg Leu Gln Lys Val Arg Val Thr
Arg Val Gln Gly Thr Phe Ser 165 170 175Asn Ala Asp Glu Gly Asp Ser
Ala Thr Val Asp Ser Ser Pro Ser Pro 180 185 190Ala Pro Thr Glu Ala
Phe Leu Val Ser Leu Ala Val Asp Val Pro Met 195 200 205Ala Glu Arg
Val Val Phe Ala Ala Glu His Gly Thr Ile Trp Leu Ser 210 215 220Asn
Glu Pro Leu Ser Ser Asn Glu Ala Gly Ala Ser Val Val Ser Pro225 230
235 240Glu Gly Val Phe Arg 24534245PRTRhodococcus equi 34Leu Arg
Thr Arg Ile Ile Ala Ala Ile Cys Ala Ile Val Leu Ala Val1 5 10 15Ala
Gly Thr Leu Ala Leu Ile Ser Tyr Val Arg Gly Ala Asp Ala Arg 20 25
30Ala Leu Ala Gly Thr Arg Thr Val Asp Val Leu Val Ala Asp Gln Thr
35 40 45Ile Pro Lys Asn Thr Pro Ala Asp Ser Leu Val Gly Met Val Val
Val 50 55 60Lys Lys Leu Pro Glu Met Ala Val Leu Pro Glu Arg Val Thr
Ser Leu65 70 75 80Asp Gln Leu Ser Gly Lys Val Ala Leu Thr Asp Leu
Leu Pro Gly Glu 85 90 95Gln Leu Val Ser Ala Arg Phe Ala Asp Pro Ala
Thr Ala Arg Ser Gln 100 105 110Asp Gln Gly Gly Ile Pro Glu Gly Met
Gln Glu Val Thr Val Leu Leu 115 120 125Glu Pro Gln Arg Ala Leu Gly
Gly His Ile Ala Pro Gly Asp Thr Val 130 135 140Gly Val Phe Met Ser
Phe Ser Pro Pro Val Lys Asn Tyr Glu Thr His145 150 155 160Leu Arg
Leu Gln Lys Val Arg Val Thr Arg Val Gln Gly Thr Phe Ser 165 170
175Asn Ala Asp Glu Gly Asp Ser Ala Thr Val Asp Ser Ser Pro Ser Pro
180 185 190Ala Pro Thr Glu Ala Phe Leu Val Ser Leu Ala Val Asp Val
Pro Met 195 200 205Ala Glu Arg Val Val Phe Ala Ala Glu His Gly Thr
Ile Trp Leu Ser 210 215 220Asn Glu Pro Leu Ser Ser Asn Glu Ala Gly
Ala Ser Val Val Ser Pro225 230 235 240Glu Gly Val Phe Arg
24535245PRTRhodococcus equi 35Leu Arg Thr Arg Ile Ile Ala Ala Ile
Cys Ala Ile Val Leu Ala Val1 5 10 15Ala Gly Thr Leu Ala Leu Ile Ser
Tyr Val Arg Gly Ala Asp Ala Arg 20 25 30Ala Leu Ala Gly Thr Arg Thr
Val Asp Val Leu Val Ala Asp Gln Thr 35 40 45Ile Pro Lys Asn Thr Pro
Ala Asp Ser Leu Val Gly Met Val Val Val 50 55 60Lys Lys Leu Pro Glu
Met Ala Val Leu Pro Asp Arg Val Thr Ser Leu65 70 75 80Asp Gln Leu
Ser Gly Lys Val Ala Leu Thr Asp Leu Leu Pro Gly Glu 85 90 95Gln Leu
Val Ser Ala Arg Phe Val Asp Pro Ala Thr Ala Arg Ser Gln 100 105
110Asp Gln Gly Gly Ile Pro Glu Gly Met Gln Glu Val Thr Val Leu Leu
115 120 125Glu Pro Gln Arg Ala Leu Gly Gly His Ile Ala Ser Gly Asp
Thr Val 130 135 140Gly Val Phe Met Ser Phe Ser Pro Pro Val Lys Asn
Tyr Glu Thr His145 150 155 160Leu Arg Leu Gln Lys Val Arg Val Thr
Arg Val Gln Gly Thr Phe Ser 165 170 175Asn Ala Asp Glu
Gly Asp Ser Ala Thr Val Asp Ser Ser Pro Ser Pro 180 185 190Ala Pro
Thr Glu Ala Phe Leu Val Ser Leu Ala Val Asp Val Pro Met 195 200
205Ala Glu Arg Val Val Phe Ala Ala Glu His Gly Thr Ile Trp Leu Ser
210 215 220Asn Glu Pro Leu Ser Ser Asn Glu Ala Gly Ala Ser Val Val
Ser Pro225 230 235 240Glu Gly Val Phe Arg 24536399PRTRhodococcus
equi 36Met Ser Arg Ile Val Leu Leu Thr Asp Arg Asp Asp Phe Ala Arg
Arg1 5 10 15Val Tyr His Ala Ala Asp Gly Asn Leu Leu Val Leu Pro Ala
Gln Pro 20 25 30Val Pro Arg Gly Pro Ala Gln Leu Val Gly Leu Gly Val
Thr Val Gln 35 40 45Pro Glu Val Leu Val Leu Gly Pro Asp Val Pro Glu
Val Glu Gly Leu 50 55 60Ser Leu Ala Gly Arg Ile Asp His Ser Thr Pro
Gly Thr Thr Val Val65 70 75 80Leu Ala Ser Asp Ala Gly Thr Asp Val
Trp Leu Arg Ala Met Arg Ala 85 90 95Gly Val Arg Asp Val Met Ser Pro
Glu Ala Glu Ile Ala Asp Val Arg 100 105 110Ala Val Leu Asp Arg Ala
Gly Gln Ala Ala Leu Ala Arg Arg Gln Gly 115 120 125Ala Ser Ala Pro
Ala Glu Gln His Ala Val Gln Gly Lys Val Ile Val 130 135 140Val Ala
Ser Pro Lys Gly Gly Thr Gly Lys Thr Thr Val Ala Thr Asn145 150 155
160Leu Ala Val Gly Leu Ala Ala Ala Ala Pro His Ser Thr Val Leu Val
165 170 175Asp Leu Asp Val Gln Phe Gly Asp Val Ala Ser Ala Leu Gln
Leu Val 180 185 190Pro Glu His Cys Leu Thr Asp Ala Val Ala Gly Pro
Ala Ser Gln Asp 195 200 205Met Ile Val Leu Lys Thr Val Leu Thr Pro
His Ser Thr Gly Leu His 210 215 220Ala Leu Cys Gly Ser Asp Ser Pro
Ala Ala Gly Asp Ser Ile Thr Gly225 230 235 240Glu Gln Val Ser Thr
Leu Leu Thr Gln Leu Ala Ala Glu Phe Arg Tyr 245 250 255Val Val Val
Asp Thr Ala Pro Gly Leu Leu Glu His Thr Leu Ala Ala 260 265 270Leu
Asp Leu Ala Thr Asp Val Val Leu Val Ser Gly Met Asp Val Pro 275 280
285Ser Val Arg Gly Met His Lys Glu Leu Gln Leu Leu Ala Glu Leu Asn
290 295 300Leu Gly Pro Val Val Arg His Val Val Leu Asn Phe Ala Asp
Arg Arg305 310 315 320Glu Gly Leu Thr Val Gln Asp Ile Gln Asn Thr
Ile Gly Val Pro Ala 325 330 335Asp Ile Val Ile Lys Arg Ser Lys Ala
Val Ala Leu Ser Thr Asn Arg 340 345 350Gly Val Pro Leu Leu Gln Asn
Pro Gly Arg Asp Arg Thr Ala Lys Glu 355 360 365Leu Trp Arg Leu Val
Gly Arg Ile Asp Pro Ala Pro Asp Thr Thr Lys 370 375 380Gly Gly Arg
Ala Arg His Arg Ala Ala Glu Ala Val Gly Ala Lys385 390
39537398PRTRhodococcus equi 37Met Ser Arg Ile Val Leu Leu Thr Asp
Arg Asp Asp Ala Arg Arg Val1 5 10 15Tyr His Ala Ala Asp Gly Asn Leu
Leu Val Leu Pro Ala Gln Pro Val 20 25 30Pro Arg Gly Pro Ala Gln Leu
Val Gly Leu Gly Val Thr Val Gln Pro 35 40 45Asp Val Leu Val Leu Gly
Pro Asp Val Pro Glu Val Glu Gly Leu Ser 50 55 60Leu Ala Gly Arg Ile
Asp His Ser Thr Pro Gly Thr Thr Val Val Leu65 70 75 80Ala Ser Asp
Ala Gly Thr Asp Val Trp Leu Arg Ala Met Arg Ala Gly 85 90 95Val Arg
Asp Val Met Ser Pro Glu Ala Glu Ile Ala Asp Val Arg Ala 100 105
110Val Leu Asp Arg Ala Gly Gln Ala Ala Leu Ala Arg Arg Gln Gly Ala
115 120 125Ser Ala Pro Ala Glu Gln His Ala Val Gln Gly Lys Val Ile
Val Val 130 135 140Ala Ser Pro Lys Gly Gly Thr Gly Lys Thr Thr Val
Ala Thr Asn Leu145 150 155 160Ala Val Gly Leu Ala Ala Ala Ala Pro
His Ser Thr Val Leu Val Asp 165 170 175Leu Asp Val Gln Phe Gly Asp
Val Ala Ser Ala Leu Gln Leu Val Pro 180 185 190Glu His Cys Leu Thr
Asp Ala Val Ala Ser Pro Ala Ser Gln Asp Met 195 200 205Ile Val Leu
Lys Thr Val Leu Thr Pro His Ser Thr Gly Leu His Ala 210 215 220Leu
Cys Gly Ser Asp Ser Pro Ala Ala Gly Asp Ser Ile Thr Gly Glu225 230
235 240Gln Val Ser Thr Leu Leu Thr Gln Leu Ala Ala Glu Phe Arg Tyr
Val 245 250 255Val Val Asp Thr Ala Pro Gly Leu Leu Glu His Thr Leu
Ala Ala Leu 260 265 270Asp Leu Ala Thr Asp Val Val Leu Val Ser Gly
Met Asp Val Pro Ser 275 280 285Val Arg Gly Met His Lys Glu Leu Gln
Leu Leu Thr Glu Leu Asn Leu 290 295 300Gly Pro Val Val Arg His Val
Val Leu Asn Phe Ala Asp Arg Arg Glu305 310 315 320Gly Leu Thr Val
Gln Asp Ile Gln Asn Thr Ile Gly Val Pro Ala Asp 325 330 335Ile Val
Ile Lys Arg Ser Lys Ala Val Ala Leu Ser Thr Asn Arg Gly 340 345
350Val Pro Leu Leu Gln Asn Pro Gly Arg Asp Arg Thr Ala Lys Glu Leu
355 360 365Trp Arg Leu Val Gly Arg Ile Asp Pro Ala Pro Asp Thr Ala
Lys Gly 370 375 380Gly Arg Ala Arg His Arg Ala Ala Glu Ala Val Gly
Ala Lys385 390 39538399PRTRhodococcus equi 38Met Ser Arg Ile Val
Leu Leu Thr Asp Arg Asp Asp Phe Ala Arg Arg1 5 10 15Val Tyr His Ala
Ala Asp Gly Asn Leu Leu Val Leu Pro Ala Gln Pro 20 25 30Val Pro Arg
Gly Pro Ala Gln Leu Val Gly Leu Gly Val Thr Val Gln 35 40 45Pro Asp
Val Leu Val Leu Gly Pro Asp Val Pro Glu Val Glu Gly Leu 50 55 60Ser
Leu Ala Gly Arg Ile Asp His Ser Thr Pro Gly Thr Thr Val Val65 70 75
80Leu Ala Ser Asp Ala Gly Thr Asp Val Trp Leu Arg Ala Met Arg Ala
85 90 95Gly Val Arg Asp Val Met Ser Pro Glu Ala Glu Ile Ala Asp Val
Arg 100 105 110Ala Val Leu Asp Arg Ala Gly Gln Ala Ala Leu Ala Arg
Arg Gln Gly 115 120 125Ala Ser Ala Pro Ala Glu Gln His Ala Val Gln
Gly Lys Val Ile Val 130 135 140Val Ala Ser Pro Lys Gly Gly Thr Gly
Lys Thr Thr Val Ala Thr Asn145 150 155 160Leu Ala Val Gly Leu Ala
Ala Ala Ala Pro His Ser Thr Val Leu Val 165 170 175Asp Leu Asp Val
Gln Phe Gly Asp Val Ala Ser Ala Leu Gln Leu Val 180 185 190Pro Glu
His Cys Leu Thr Asp Ala Val Ala Ser Pro Ala Ser Gln Asp 195 200
205Met Ile Val Leu Lys Thr Val Leu Thr Pro His Ser Thr Gly Leu His
210 215 220Ala Leu Cys Gly Ser Asp Ser Pro Ala Ala Gly Asp Ser Ile
Thr Gly225 230 235 240Glu Gln Val Ser Thr Leu Leu Thr Gln Leu Ala
Ala Glu Phe Arg Tyr 245 250 255Val Val Val Asp Thr Ala Pro Gly Leu
Leu Glu His Thr Leu Ala Ala 260 265 270Leu Asp Leu Ala Thr Asp Val
Val Leu Val Ser Gly Met Asp Val Pro 275 280 285Ser Val Arg Gly Met
His Lys Glu Leu Gln Leu Leu Thr Glu Leu Asn 290 295 300Leu Gly Pro
Val Val Arg His Val Val Leu Asn Phe Ala Asp Arg Arg305 310 315
320Glu Gly Leu Thr Val Gln Asp Ile Gln Asn Thr Ile Gly Val Pro Ala
325 330 335Asp Ile Val Ile Lys Arg Ser Lys Ala Val Ala Leu Ser Thr
Asn Arg 340 345 350Gly Val Pro Leu Leu Gln Asn Pro Gly Arg Asp Arg
Thr Ala Lys Glu 355 360 365Leu Trp Arg Leu Val Gly Arg Ile Asp Pro
Ala Pro Asp Thr Ala Lys 370 375 380Gly Gly Arg Ala Arg His Arg Ala
Ala Glu Ala Val Gly Ala Lys385 390 39539465PRTRhodococcus equi
39Met Arg Leu Ser Gln Arg Leu Glu Ala Val Arg Gly Ala Ala Pro Val1
5 10 15Glu Ala Ala Ala Pro Ile Pro Pro Gly Lys Gln Gly Lys Ala Lys
Thr 20 25 30Ser Leu Pro Pro Ala Asp Ala Leu Ala Glu Leu Lys Asp Arg
Ala Ser 35 40 45Ala Ala Leu Tyr Thr Arg Ile Gly Thr Arg Phe Asn Asp
Ser Ser Leu 50 55 60Ser Glu Glu Gln Leu His Leu Leu Val Arg Glu Glu
Leu Ala Glu Ile65 70 75 80Val Glu Gln Glu Thr Thr Pro Leu Thr Phe
Asp Glu Arg Gln Arg Leu 85 90 95Leu Arg Glu Val Ala Asp Glu Val Leu
Gly His Gly Pro Leu Gln Arg 100 105 110Leu Leu Glu Asp Pro Ser Val
Thr Glu Ile Met Val Asn Ser His Asp 115 120 125Met Val Tyr Val Glu
Arg Asp Gly Thr Leu Val Arg Ser Ser Ala Arg 130 135 140Phe Ala Asp
Glu Ala His Leu Arg Arg Val Ile Glu Arg Ile Val Ser145 150 155
160Ala Val Gly Arg Arg Ile Asp Glu Ser Ser Pro Leu Val Asp Ala Arg
165 170 175Leu Ala Asp Gly Ser Arg Val Asn Ala Val Ile Pro Pro Leu
Ala Phe 180 185 190Asn Gly Ser Ser Leu Thr Ile Arg Lys Phe Ser Lys
Asp Pro Phe Gln 195 200 205Val Asp Asp Leu Ile Ala Phe Gly Thr Leu
Ser His Glu Met Ala Glu 210 215 220Leu Leu Asp Ala Cys Val Gln Ala
Arg Leu Asn Val Ile Val Ser Gly225 230 235 240Gly Thr Gly Thr Gly
Lys Thr Thr Leu Leu Asn Val Leu Ser Ser Phe 245 250 255Ile Pro Glu
Gly Glu Arg Ile Val Thr Ile Glu Asp Ala Val Glu Leu 260 265 270Gln
Leu Gln Gln Asp His Val Val Arg Leu Glu Ser Arg Pro Pro Asn 275 280
285Ile Glu Gly Lys Gly Ala Val Thr Ile Arg Asp Leu Val Arg Asn Ser
290 295 300Leu Arg Met Arg Pro Asp Arg Ile Val Val Gly Glu Cys Arg
Gly Gly305 310 315 320Glu Ser Leu Asp Met Leu Gln Ala Met Asn Thr
Gly His Asp Gly Ser 325 330 335Leu Ser Thr Val His Ala Asn Ser Pro
Arg Asp Ala Ile Ala Arg Leu 340 345 350Glu Thr Leu Val Leu Met Ala
Gly Met Asp Leu Pro Leu Arg Ala Ile 355 360 365Arg Glu Gln Ile Ala
Ser Ala Val Asp Val Ile Val Gln Leu Thr Arg 370 375 380Leu Arg Asp
Gly Thr Arg Arg Val Thr His Val Thr Glu Val Gln Gly385 390 395
400Met Glu Gly Glu Ile Val Thr Leu Gln Asp Ala Phe Leu Phe Asp Tyr
405 410 415Ser Ala Gly Val Asp Ala Arg Gly Arg Phe Leu Gly Arg Pro
Gln Pro 420 425 430Thr Gly Val Arg Pro Arg Phe Thr Asp Lys Phe Arg
Asp Leu Gly Ile 435 440 445Ala Leu Ser Pro Ser Val Phe Gly Val Gly
Glu Pro Ser Arg Gly Arg 450 455 460Ala46540465PRTRhodococcus equi
40Met Arg Leu Ser Gln Arg Leu Glu Ala Val Arg Gly Ala Ala Pro Val1
5 10 15Glu Ala Ala Ala Pro Ile Pro Pro Gly Lys Gln Gly Lys Ala Lys
Thr 20 25 30Ser Leu Pro Pro Ala Asp Ala Leu Ala Glu Leu Lys Asp Arg
Ala Ser 35 40 45Ala Ala Leu Tyr Thr Arg Ile Gly Thr Arg Phe Asn Asp
Ser Ser Leu 50 55 60Ser Glu Glu Gln Leu His Leu Leu Val Arg Glu Glu
Leu Ala Glu Ile65 70 75 80Val Glu Gln Glu Thr Thr Pro Leu Thr Phe
Asp Glu Arg Gln Arg Leu 85 90 95Leu Arg Glu Val Ala Asp Glu Val Leu
Gly His Gly Pro Leu Gln Arg 100 105 110Leu Leu Glu Asp Pro Ser Val
Thr Glu Ile Met Val Asn Ser His Asp 115 120 125Met Val Tyr Val Glu
Arg Asp Gly Thr Leu Val Arg Ser Ser Ala Arg 130 135 140Phe Ala Asp
Glu Ala His Leu Arg Arg Val Ile Glu Arg Ile Val Ser145 150 155
160Ala Val Gly Arg Arg Ile Asp Glu Ser Ser Pro Leu Val Asp Ala Arg
165 170 175Leu Ala Asp Gly Ser Arg Val Asn Ala Val Ile Pro Pro Leu
Ala Phe 180 185 190Asn Gly Ser Ser Leu Thr Ile Arg Lys Phe Ser Lys
Asp Pro Phe Gln 195 200 205Val Asp Asp Leu Ile Ala Phe Gly Thr Leu
Ser His Glu Met Ala Glu 210 215 220Leu Leu Asp Ala Cys Val Gln Ala
Arg Leu Asn Val Ile Val Ser Gly225 230 235 240Gly Thr Gly Thr Gly
Lys Thr Thr Leu Leu Asn Val Leu Ser Ser Phe 245 250 255Ile Pro Glu
Gly Glu Arg Ile Val Thr Ile Glu Asp Ala Val Glu Leu 260 265 270Gln
Leu Gln Gln Asp His Val Val Arg Leu Glu Ser Arg Pro Pro Asn 275 280
285Ile Glu Gly Lys Gly Ala Val Thr Ile Arg Asp Leu Val Arg Asn Ser
290 295 300Leu Arg Met Arg Pro Asp Arg Ile Val Val Gly Glu Cys Arg
Gly Gly305 310 315 320Glu Ser Leu Asp Met Leu Gln Ala Met Asn Thr
Gly His Asp Gly Ser 325 330 335Leu Ser Thr Val His Ala Asn Ser Pro
Arg Asp Ala Ile Ala Arg Leu 340 345 350Glu Thr Leu Val Leu Met Ala
Gly Met Asp Leu Pro Leu Arg Ala Ile 355 360 365Arg Glu Gln Ile Ala
Ser Ala Val Asp Val Ile Val Gln Leu Thr Arg 370 375 380Leu Arg Asp
Gly Thr Arg Arg Val Thr His Val Thr Glu Val Gln Gly385 390 395
400Met Glu Gly Glu Ile Val Thr Leu Gln Asp Ala Phe Leu Phe Asp Tyr
405 410 415Ser Ala Gly Val Asp Ala Arg Gly Arg Phe Leu Gly Arg Pro
Gln Pro 420 425 430Thr Gly Val Arg Pro Arg Phe Thr Asp Lys Phe Arg
Asp Leu Gly Ile 435 440 445Ala Leu Ser Pro Ser Val Phe Gly Val Gly
Glu Pro Ser Arg Gly Arg 450 455 460Ala46541465PRTRhodococcus equi
41Met Arg Leu Ser Gln Arg Leu Glu Ala Val Arg Gly Ala Ala Pro Val1
5 10 15Glu Ala Ala Ala Pro Ile Pro Pro Gly Lys Gln Gly Lys Ala Lys
Thr 20 25 30Ser Leu Pro Pro Ala Asp Ala Leu Ala Glu Leu Lys Asp Arg
Ala Ser 35 40 45Ala Ala Leu Tyr Thr Arg Ile Gly Thr Arg Phe Asn Asp
Ser Ser Leu 50 55 60Ser Glu Glu Gln Leu His Leu Leu Val Arg Glu Glu
Leu Ala Glu Ile65 70 75 80Val Glu Gln Glu Thr Thr Pro Leu Thr Phe
Asp Glu Arg Gln Arg Leu 85 90 95Leu Arg Glu Val Ala Asp Glu Val Leu
Gly His Gly Pro Leu Gln Arg 100 105 110Leu Leu Glu Asp Pro Ser Val
Thr Glu Ile Met Val Asn Ser His Asp 115 120 125Met Val Tyr Val Glu
Arg Asp Gly Thr Leu Val Arg Ser Ser Ala Arg 130 135 140Phe Ala Asp
Glu Ala His Leu Arg Arg Val Ile Glu Arg Ile Val Ser145 150 155
160Ala Val Gly Arg Arg Ile Asp Glu Ser Ser Pro Leu Val Asp Ala Arg
165 170 175Leu Ala Asp Gly Ser Arg Val Asn Ala Val Ile Pro Pro Leu
Ala Phe 180 185 190Asn Gly Ser Ser Leu Thr Ile Arg Lys Phe Ser Lys
Asp Pro Phe Gln 195 200 205Val Asp Asp Leu Ile Ala Phe Gly Thr Leu
Ser His Glu Met Ala Glu 210 215 220Leu Leu Asp Ala Cys Val Gln Ala
Arg Leu Asn Val Ile Val Ser Gly225 230 235 240Gly Thr Gly Thr Gly
Lys Thr Thr Leu Leu Asn Val Leu Ser Ser Phe 245 250 255Ile Pro Glu
Gly Glu Arg Ile Val Thr Ile Glu Asp Ala Val Glu Leu 260 265 270Gln
Leu Gln Gln Asp His Val Val Arg Leu
Glu Ser Arg Pro Pro Asn 275 280 285Ile Glu Gly Lys Gly Ala Val Thr
Ile Arg Asp Leu Val Arg Asn Ser 290 295 300Leu Arg Met Arg Pro Asp
Arg Ile Val Val Gly Glu Cys Arg Gly Gly305 310 315 320Glu Ser Leu
Asp Met Leu Gln Ala Met Asn Thr Gly His Asp Gly Ser 325 330 335Leu
Ser Thr Val His Ala Asn Ser Pro Arg Asp Ala Ile Ala Arg Leu 340 345
350Glu Thr Leu Val Leu Met Ala Gly Met Asp Leu Pro Leu Arg Ala Ile
355 360 365Arg Glu Gln Ile Ala Ser Ala Val Asp Val Ile Val Gln Leu
Thr Arg 370 375 380Leu Arg Asp Gly Thr Arg Arg Val Thr His Val Thr
Glu Val Gln Gly385 390 395 400Met Glu Gly Glu Ile Val Thr Leu Gln
Asp Ala Phe Leu Phe Asp Tyr 405 410 415Ser Ala Gly Val Asp Ala Arg
Gly Arg Phe Leu Gly Arg Pro Gln Pro 420 425 430Thr Gly Val Arg Pro
Arg Phe Thr Asp Lys Phe Arg Asp Leu Gly Ile 435 440 445Ala Leu Ser
Pro Ser Val Phe Gly Val Gly Glu Pro Ser Arg Gly Arg 450 455
460Ala46542623PRTRhodococcus equi 42Met Ser Arg Cys Val Val Ala Val
Val Leu Ala Leu Gly Ala Gly Val1 5 10 15Leu Gly Ile Pro Ala Val Ala
Ala Ala Ala Glu Thr Glu Ala Val Gln 20 25 30Val Ser Ala Val Asp Thr
Thr Arg Phe Pro Asp Ile Glu Val Ser Ile 35 40 45Leu Ala Pro Pro Gly
Ile Glu Gly Gln Ala Ile Asp Pro Gly Thr Phe 50 55 60Ala Leu Thr Glu
Gly Gly Val Pro Arg Glu Ile Glu Val Arg Gln Gln65 70 75 80Pro Gly
Ser Glu Gln Asp Ile Val Leu Ala Ile Asp Val Ser Gly Gly 85 90 95Met
Ser Gly Pro Ala Leu Asp Asp Val Lys Arg Ala Ala Ser Asp Phe 100 105
110Val Arg Gln Ala Pro Thr Gly Ala His Ile Gly Ile Val Ala Ile Ser
115 120 125Ser Thr Pro Gln Val Leu Ser Glu Leu Thr Thr Asp Ser Glu
Asp Leu 130 135 140Leu Arg Arg Ile Asp Gly Leu Lys Ala Gly Gly Asn
Ser Ala Ile Ala145 150 155 160Asp Ser Val Val Thr Ala Ala Glu Met
Leu Glu Arg Gly Glu Ala Ala 165 170 175Asn Asn Ile Leu Leu Leu Leu
Thr Asp Gly Ala Asp Thr Ser Ser Ala 180 185 190His Ser Met Ser Glu
Leu Pro Ser Val Leu Ser Arg Ser Arg Ala Ser 195 200 205Leu Tyr Ala
Val Gln Met Ser Thr Pro Glu Thr Asn Ser Ala Leu Leu 210 215 220Gln
Gln Val Ala Arg Glu Ser Arg Gly Gln Tyr Ala Ser Ala Gly Asp225 230
235 240Thr Ala Ala Leu Gly Ala Ile Tyr Gln Ser Ala Ala Arg Ala Leu
Gly 245 250 255Asn Leu Tyr Val Val Arg Tyr Arg Ser Glu Ala Asn Gly
Asp Thr Gln 260 265 270Val Val Ala Ser Val Arg Ser Gly Ala Ala Gly
Arg Val Ser Asp Pro 275 280 285Phe Pro Val Thr Leu Pro Gly Val Val
Pro Thr Pro Ser Val Val Ala 290 295 300Gly Thr Val Asp Gly Phe Phe
Thr Ser Ser Thr Gly Leu Val Ile Gly305 310 315 320Leu Leu Ala Cys
Tyr Ser Ala Leu Ala Gly Gly Val Leu Ala Val Ala 325 330 335Gly Arg
Ala Pro Ala Arg Ile Ser Ala Ala Arg Arg Gly Arg Gln Asp 340 345
350Gly Arg Asp Ser Met Leu Ser Arg Phe Ala Glu Arg Leu Val Gln Trp
355 360 365Ile Asp Gln Asn Leu Arg Arg Arg Gly Arg Ile Ala Ala Arg
Thr Gln 370 375 380Ala Leu Gln Glu Ala Gly Leu Lys Leu Arg Pro Gly
Asp Phe Ile Ala385 390 395 400Leu Val Gly Ala Ala Ala Ile Thr Ala
Ala Ala Ile Gly Leu Leu Ala 405 410 415Ser Gly Ile Val Ala Ala Leu
Leu Leu Ala Ala Ile Thr Val Gly Leu 420 425 430Ser Arg Ile Tyr Leu
Arg Val Met Ala Gly Arg Arg Arg Ala Ala Phe 435 440 445Ala Asp Gln
Leu Asp Asp Ser Leu Gln Leu Leu Ala Ser Asn Leu Arg 450 455 460Ala
Gly His Ser Met Leu Arg Ala Leu Asp Ser Leu Ser Arg Glu Ala465 470
475 480Glu Val Pro Thr Ser Glu Glu Phe Ala Arg Ile Val Asn Glu Thr
Arg 485 490 495Val Gly Arg Asp Leu Asn Glu Ser Leu Asp Asp Val Ala
Arg Arg Met 500 505 510Arg Ser Asp Asp Phe Asn Trp Ile Ala Gln Ala
Ile Ala Ile Asn Arg 515 520 525Glu Val Gly Gly Asp Leu Ala Glu Val
Leu Asp Gln Val Gly Asn Thr 530 535 540Ile Arg Glu Arg Asn Gln Ile
Arg Arg Gln Val Lys Ala Leu Ala Ala545 550 555 560Glu Gly Lys Leu
Ser Ala Tyr Val Leu Met Ala Leu Pro Phe Gly Leu 565 570 575Thr Ala
Phe Leu Leu Val Ser Asn Pro Asp Tyr Leu Ser Lys Leu Thr 580 585
590Gly Ser Ala Ile Gly Tyr Val Met Ile Ala Val Gly Leu Val Met Leu
595 600 605Thr Val Gly Gly Leu Trp Met Asn Lys Val Val Ser Val Lys
Phe 610 615 62043621PRTRhodococcus equi 43Met Ser Arg Cys Val Val
Ala Val Val Leu Ala Leu Gly Ala Gly Val1 5 10 15Leu Gly Ile Pro Ala
Val Ala Ala Ala Ala Glu Thr Glu Ala Val Gln 20 25 30Val Ser Ala Val
Asp Thr Thr Arg Phe Pro Asp Ile Glu Val Ser Ile 35 40 45Leu Ala Pro
Pro Gly Ile Glu Gly Gln Ala Ile Asp Pro Gly Thr Phe 50 55 60Ala Leu
Thr Glu Gly Gly Val Pro Arg Glu Ile Glu Val Arg Gln Gln65 70 75
80Pro Gly Ser Glu Gln Asp Ile Val Leu Ala Ile Asp Val Ser Gly Gly
85 90 95Met Ser Gly Pro Ala Leu Asp Asp Val Lys Arg Ala Ala Ser Asp
Phe 100 105 110Val Arg Gln Ala Pro Thr Gly Ala His Ile Gly Ile Val
Ala Ile Ser 115 120 125Ser Thr Pro Gln Val Leu Ser Glu Leu Thr Thr
Asp Ser Glu Asp Leu 130 135 140Leu Arg Arg Ile Asp Gly Leu Lys Ala
Gly Gly Asn Ser Ala Ile Ala145 150 155 160Asp Ser Val Val Thr Ala
Ala Glu Met Leu Glu Arg Gly Glu Ala Ala 165 170 175Asn Asn Ile Leu
Leu Leu Leu Thr Asp Gly Ala Asp Thr Ser Ser Ala 180 185 190His Ser
Met Ser Glu Leu Pro Ser Val Leu Ser Arg Ser Arg Ala Ser 195 200
205Leu Tyr Ala Val Gln Met Ser Thr Pro Glu Thr Asn Ser Ala Leu Leu
210 215 220Gln Gln Val Ala Arg Glu Ser Arg Gly Gln Tyr Ala Ser Ala
Gly Asp225 230 235 240Thr Ala Ala Leu Gly Ala Ile Tyr Gln Ser Ala
Ala Arg Ala Leu Gly 245 250 255Asn Leu Tyr Val Val Arg Tyr Arg Ser
Glu Ala Asn Gly Asp Thr Gln 260 265 270Val Val Ala Ser Val Arg Ser
Gly Ala Ala Gly Arg Val Ser Asp Pro 275 280 285Phe Pro Val Thr Leu
Pro Gly Val Val Pro Thr Pro Ser Val Val Ala 290 295 300Gly Thr Val
Asp Gly Phe Phe Thr Ser Ser Thr Gly Leu Val Ile Gly305 310 315
320Leu Leu Ala Cys Tyr Ser Ala Leu Ala Gly Leu Ala Val Ala Gly Arg
325 330 335Gly Pro Ala Arg Ile Ser Ala Ala Arg Arg Gly Arg Gln Asp
Gly Arg 340 345 350Asp Ser Met Leu Ser Arg Phe Ala Glu Arg Leu Val
Gln Trp Ile Asp 355 360 365Gln Asn Leu Arg Arg Arg Gly Arg Ile Ala
Ala Arg Thr Gln Ala Leu 370 375 380Gln Glu Ala Gly Leu Lys Leu Arg
Pro Gly Asp Phe Ile Ala Leu Val385 390 395 400Gly Ala Ala Ala Ile
Thr Ala Ala Ala Ile Gly Leu Leu Ala Ser Gly 405 410 415Ile Val Ala
Ala Leu Leu Leu Ala Ala Ile Thr Val Gly Leu Ser Arg 420 425 430Ile
Tyr Leu Arg Val Met Ala Gly Arg Arg Arg Ala Ala Phe Ala Asp 435 440
445Gln Leu Asp Asp Ser Leu Gln Leu Leu Ala Ser Asn Leu Arg Ala Gly
450 455 460His Ser Met Leu Arg Ala Leu Asp Ser Leu Ser Arg Glu Ala
Glu Val465 470 475 480Pro Thr Ser Glu Glu Phe Ala Arg Ile Val Asn
Glu Thr Arg Val Gly 485 490 495Arg Asp Leu Asn Glu Ser Leu Asp Asp
Val Ala Arg Arg Met Arg Ser 500 505 510Asp Asp Phe Asn Trp Ile Ala
Gln Ala Ile Ala Ile Asn Arg Glu Val 515 520 525Gly Gly Asp Leu Ala
Glu Val Leu Asp Gln Val Gly Asn Thr Ile Arg 530 535 540Glu Arg Asn
Gln Ile Arg Arg Gln Val Lys Ala Leu Ala Ala Glu Gly545 550 555
560Lys Leu Ser Ala Tyr Val Leu Met Ala Leu Pro Phe Gly Leu Thr Ala
565 570 575Phe Leu Leu Val Ser Asn Pro Asp Tyr Leu Ser Lys Leu Thr
Gly Ser 580 585 590Ala Ile Gly Tyr Val Met Ile Ala Val Gly Leu Val
Met Leu Thr Val 595 600 605Gly Gly Leu Trp Met Asn Lys Val Val Ser
Val Lys Phe 610 615 62044620PRTRhodococcus equi 44Met Ser Arg Cys
Val Val Ala Val Val Leu Ala Leu Gly Ala Gly Val1 5 10 15Leu Gly Ile
Pro Ala Val Ala Ala Ala Glu Ala Val Gln Val Ser Ala 20 25 30Val Asp
Thr Thr Arg Phe Pro Asp Ile Glu Val Ser Ile Leu Ala Pro 35 40 45Pro
Gly Ile Glu Gly Gln Ala Ile Asp Pro Gly Thr Phe Ala Leu Thr 50 55
60Glu Gly Gly Val Pro Arg Glu Ile Glu Val Arg Gln Gln Pro Gly Ser65
70 75 80Glu Gln Asp Ile Val Leu Ala Ile Asp Val Ser Gly Gly Met Ser
Gly 85 90 95Pro Ala Leu Asp Asp Val Lys Arg Ala Ala Ser Asp Phe Val
Arg Gln 100 105 110Ala Pro Thr Gly Ala His Ile Gly Ile Val Ala Ile
Ser Ser Thr Pro 115 120 125Gln Val Leu Ser Glu Leu Thr Thr Asp Ser
Glu Asp Leu Leu Arg Arg 130 135 140Ile Asp Gly Leu Lys Ala Gly Gly
Asn Ser Ala Ile Ala Asp Ser Val145 150 155 160Val Thr Ala Ala Glu
Met Leu Glu Arg Gly Glu Ala Ala Asn Asn Ile 165 170 175Leu Leu Leu
Leu Thr Asp Gly Ala Asp Thr Ser Ser Ala His Ser Met 180 185 190Ser
Glu Leu Pro Ser Val Leu Ser Arg Ser Arg Ala Ser Leu Tyr Ala 195 200
205Val Gln Met Ser Thr Pro Glu Thr Asn Ser Ala Leu Leu Gln Gln Val
210 215 220Ala Arg Glu Ser Arg Gly Gln Tyr Ala Ser Ala Gly Asp Thr
Ala Ala225 230 235 240Leu Gly Ala Ile Tyr Gln Ser Ala Ala Arg Ala
Leu Gly Asn Leu Tyr 245 250 255Val Val Arg Tyr Arg Ser Glu Ala Asn
Gly Asp Thr Gln Val Val Ala 260 265 270Ser Val Arg Ser Gly Ala Ala
Gly Arg Val Ser Asp Pro Phe Pro Val 275 280 285Thr Leu Pro Gly Val
Val Pro Thr Pro Ser Val Val Ala Gly Thr Val 290 295 300Asp Gly Phe
Phe Thr Ser Ser Thr Gly Leu Val Ile Gly Leu Leu Ala305 310 315
320Cys Tyr Ser Ala Leu Ala Gly Gly Val Leu Ala Val Ala Gly Arg Ala
325 330 335Pro Ala Arg Ile Ser Ala Ala Arg Arg Gly Arg Gln Asp Gly
Arg Asp 340 345 350Ser Met Leu Ser Arg Phe Ala Glu Arg Leu Val Gln
Trp Ile Asp Gln 355 360 365Asn Leu Arg Arg Arg Gly Arg Ile Ala Ala
Arg Thr Gln Ala Leu Gln 370 375 380Glu Ala Gly Leu Lys Leu Arg Pro
Gly Asp Phe Ile Ala Leu Val Gly385 390 395 400Ala Ala Ala Ile Thr
Ala Ala Ala Ile Gly Leu Leu Ala Ser Gly Ile 405 410 415Val Ala Ala
Leu Leu Leu Ala Ala Ile Thr Val Gly Leu Ser Arg Ile 420 425 430Tyr
Leu Arg Val Met Ala Gly Arg Arg Arg Ala Ala Phe Ala Asp Gln 435 440
445Leu Asp Asp Ser Leu Gln Leu Leu Ala Ser Asn Leu Arg Ala Gly His
450 455 460Ser Met Leu Arg Ala Leu Asp Ser Leu Ser Arg Glu Ala Glu
Val Pro465 470 475 480Thr Ser Glu Glu Phe Ala Arg Ile Val Asn Glu
Thr Arg Val Gly Arg 485 490 495Asp Leu Asn Glu Ala Leu Asp Asp Val
Ala Arg Arg Met Arg Ser Asp 500 505 510Asp Phe Asn Trp Ile Ala Gln
Ala Ile Ala Ile Asn Arg Glu Val Gly 515 520 525Gly Asp Leu Ala Glu
Val Leu Asp Gln Val Gly Asn Thr Ile Arg Glu 530 535 540Arg Asn Gln
Ile Arg Arg Gln Val Lys Ala Leu Ala Ala Glu Gly Lys545 550 555
560Leu Ser Ala Tyr Val Leu Met Ala Leu Pro Phe Gly Leu Thr Ala Phe
565 570 575Leu Leu Val Ser Asn Pro Asp Tyr Leu Ser Lys Leu Thr Gly
Ser Ala 580 585 590Ile Gly Tyr Val Met Ile Ala Val Gly Leu Val Met
Leu Thr Val Gly 595 600 605Gly Leu Trp Met Asn Lys Val Val Ser Val
Lys Phe 610 615 62045296PRTRhodococcus equi 45Val Ile Pro Pro Leu
Val Leu Met Ala Ala Leu Ser Val Gly Gly Ala1 5 10 15Leu Gly Val Leu
Val Trp Leu Thr Ala Gly Ala Arg Asp Pro Glu Arg 20 25 30Gly Pro Ala
Leu Gln Asn Leu Gln Ser Gln Leu Ala Leu Pro Ile Pro 35 40 45Glu Ser
Gly Gly Ala Pro Pro Leu Ser Leu Gly Arg Phe Val Lys Leu 50 55 60Leu
Ser Pro Pro Gly Thr Met Ala Arg Leu Glu Arg Leu His Ile Leu65 70 75
80Ala Gly Arg Pro Ala Ala Trp Val Pro Glu Arg Ala Ala Met Ala Lys
85 90 95Ile Val Leu Ala Ala Ala Ala Ala Leu Leu Gly Leu Leu Ala Val
Gly 100 105 110Ala Ser Pro Gly Val Gly Arg Val Leu Phe Ala Ala Ala
Ala Val Ala 115 120 125Leu Ala Tyr Phe Val Pro Glu Leu Leu Leu Gln
Ser Arg Gly Gln Glu 130 135 140Arg Gln Ala Ala Ile Glu Leu Ala Leu
Ala Asp Thr Leu Asp Gln Met145 150 155 160Thr Ile Ala Val Glu Ala
Gly Leu Gly Phe Glu Ala Ala Met Gln Arg 165 170 175Ala Ala Lys Asn
Gly Lys Gly Pro Leu Ala Glu Glu Phe Ile Arg Thr 180 185 190Leu Gln
Asp Ile Gln Met Gly Gln Ser Arg Arg Ile Ala Tyr Leu Asp 195 200
205Leu Ala Ala Arg Thr Lys Ala Pro Asn Leu Arg Arg Phe Leu Arg Ala
210 215 220Val Ile Gln Ala Asp Glu Tyr Gly Val Ala Ile Ala Glu Val
Leu Arg225 230 235 240Thr Gln Ala Ser Glu Met Arg Leu Lys Arg Arg
Gln Ser Ala Glu Glu 245 250 255Lys Ala Met Lys Val Pro Val Lys Val
Leu Phe Pro Leu Met Thr Cys 260 265 270Ile Leu Pro Thr Ile Phe Ile
Val Ile Leu Gly Pro Ala Val Ile Asn 275 280 285Met Met Glu Val Leu
Gly Gly Met 290 29546296PRTRhodococcus equi 46Val Ile Pro Pro Leu
Val Leu Val Ala Ala Leu Ser Val Gly Gly Ala1 5 10 15Leu Gly Val Leu
Val Trp Leu Thr Ala Gly Ala Arg Asp Pro Glu Arg 20 25 30Gly Pro Ala
Leu Gln Asn Leu Gln Ser Gln Leu Ala Leu Pro Ile Pro 35 40 45Val Ser
Gly Gly Ala Pro Pro Leu Ser Leu Gly Arg Phe Val Lys Leu 50 55 60Leu
Ser Pro Pro Gly Thr Met Ala Arg Leu Glu Arg Leu His Ile Leu65 70 75
80Ala Gly Arg Pro Ala Ala Trp Val Pro Glu Arg Ala Ala Met Ala Lys
85 90 95Ile Val Leu Ala Ala Ala Ala Ala Leu Leu Gly Leu Leu Ala Val
Gly 100 105
110Ala Ser Pro Gly Val Gly Arg Val Leu Phe Ala Ala Ala Ala Val Ala
115 120 125Leu Ala Tyr Phe Val Pro Glu Leu Leu Leu Gln Ser Arg Gly
Gln Glu 130 135 140Arg Gln Ala Ala Ile Glu Leu Ala Leu Ala Asp Thr
Leu Asp Gln Met145 150 155 160Thr Ile Ala Val Glu Ala Gly Leu Gly
Phe Glu Ala Ala Met Gln Arg 165 170 175Ala Ala Lys Asn Gly Lys Gly
Pro Leu Ala Glu Glu Phe Ile Arg Thr 180 185 190Leu Gln Asp Ile Gln
Met Gly Gln Ser Arg Arg Ile Ala Tyr Leu Asp 195 200 205Leu Ala Ala
Arg Thr Lys Ala Pro Asn Leu Arg Arg Phe Leu Arg Ala 210 215 220Val
Ile Gln Ala Asp Glu Tyr Gly Val Ala Ile Ala Glu Val Leu Arg225 230
235 240Thr Gln Ala Ser Glu Met Arg Leu Lys Arg Arg Gln Ser Ala Glu
Glu 245 250 255Lys Ala Met Lys Val Pro Val Lys Val Leu Phe Pro Leu
Met Thr Cys 260 265 270Ile Leu Pro Thr Ile Phe Ile Val Ile Leu Gly
Pro Ala Val Ile Asn 275 280 285Met Met Glu Val Leu Gly Gly Met 290
29547296PRTRhodococcus equi 47Val Ile Pro Pro Leu Val Leu Met Ala
Ala Leu Ser Val Gly Gly Ala1 5 10 15Leu Gly Val Leu Val Trp Leu Thr
Ala Gly Ala Arg Asp Pro Glu Arg 20 25 30Gly Pro Ala Leu Gln Asn Leu
Gln Ser Gln Leu Ala Leu Pro Ile Pro 35 40 45Glu Ser Gly Gly Ala Pro
Pro Ile Ser Leu Gly Arg Phe Val Lys Leu 50 55 60Leu Ser Pro Pro Gly
Thr Met Ala Arg Leu Glu Arg Leu His Ile Leu65 70 75 80Ala Gly Arg
Pro Ala Ala Trp Val Pro Glu Arg Ala Ala Met Ala Lys 85 90 95Ile Val
Leu Ala Ala Ala Ala Ala Leu Leu Gly Leu Leu Ala Ala Gly 100 105
110Ala Ser Pro Gly Val Gly Arg Val Leu Phe Ala Ala Ala Ala Val Ala
115 120 125Leu Ala Tyr Phe Val Pro Glu Leu Leu Leu Gln Ser Arg Val
Gln Glu 130 135 140Arg Gln Ala Ala Ile Glu Leu Ala Leu Ala Asp Thr
Leu Asp Gln Met145 150 155 160Thr Ile Ala Val Glu Ala Gly Leu Gly
Phe Glu Ala Ala Met Gln Arg 165 170 175Ala Ala Lys Asn Gly Lys Gly
Pro Leu Ala Glu Glu Phe Ile Arg Thr 180 185 190Leu Gln Asp Ile Gln
Met Gly Gln Ser Arg Arg Ile Ala Tyr Leu Asp 195 200 205Leu Ala Ala
Arg Thr Lys Ala Pro Asn Leu Arg Arg Phe Leu Arg Ala 210 215 220Val
Ile Gln Ala Asp Glu Tyr Gly Val Ala Ile Ala Glu Val Leu Arg225 230
235 240Thr Gln Ala Ser Glu Met Arg Leu Lys Arg Arg Gln Ser Ala Glu
Glu 245 250 255Lys Ala Met Lys Val Pro Val Lys Val Leu Phe Pro Leu
Met Thr Cys 260 265 270Ile Leu Pro Thr Ile Phe Ile Val Ile Leu Gly
Pro Ala Val Ile Asn 275 280 285Met Met Glu Val Leu Gly Gly Met 290
29548789DNARhodococcus equi 48gtgatcgtcg cagcgggcgt cggcgctgcc
ctcctgggca tcctcgccgg ggcgttcgca 60aacagtgcga tcgaccgcgt gcgcctggag
accgcgtgcg ccgagccgaa gtcgaccccc 120gccaactcaa ccccgccgtc
cccctcccct acgtccgcgg tggccgcccg gatcgcgatg 180atcgacacca
tcacgcgacg acacgacatc agtgcccgcc gcgtgctcgt cgaactcgca
240actgccctcc tgttcgtcgc gatcactctc cgtctcgccg ctctcgatct
tctcccggca 300gcaccggcct atctctggtt cgccgtcgtc gggatcgccc
tcgccgtcat cgacatcgat 360tgcaaacggc tgccgaactt cctcgtcgta
ccgtcgtacc cgatcgtatt cgcctgcctg 420gcagtgggtt ccgtcgtcac
gggcgactgg tcggccctgc tgcgcgccgc gatcggtgcc 480gccgtcctgt
tcgggttcta cttcgtactc gccctgatct atccggccgg catggggttc
540ggcgacgtca aacttgccgg cgtcatcggc gccgtcctcg cctacctgtc
gtacggcaca 600ctgctcgtcg gggcgtttct cgcgttcctg gtggccgcac
tcgtcggcct gatcatcctg 660gtcacccgtc gcggacggat cgggaccacg
attcccttcg ggccgtacat gattgcggcg 720gccgtcgttg cgatcctggc
agccgatccg ctggcgcgtg cgtatctgga ctgggccgcc 780gcggcctga
78949789DNARhodococcus equi 49gtgatcgtcg cagcgggcgt cggcgccgca
ctcctgggca tccttgccgg ggcattcgca 60aacagtgcga tcgaccgcgt gcgcctggag
accgcgtgcg ccgagccgag ggcgaccccc 120accggctcaa ccccgccgcc
cccctcccct acgtccgcgg tagccacccg gatcgcgatg 180atcgacacca
tcacgcgacg acgcgacatc agtgcccgcc gcatgctcgt cgaactcgca
240acggccctcc tgttcgtcgc gatcactctc cgtctcgccg ctctcgatct
tctcccggca 300gcaccggcct atctctggtt cgccgtcatc gggatcgccc
tcgccgtcat cgacatcgat 360tgcaaacggc tgccgaactt cctcgtcgta
ccgtcgtacc cgatcgtatt cgcctgcctg 420gcagtgggtt ccgtcgtcac
gggcgactgg tcggccctgc tgcgcgccgc gatcggtgcc 480gccgtcctgt
tcgggttcta cttcgtactc gccctgatct atccggccgg catggggttc
540ggcgacgtca aacttgccgg cgtcatcggc gccgtcctcg cctacctgtc
gtacggcaca 600ctgctcgtcg gggcgtttct cgcgttcctg gtggccgcac
tcgtgggcct catcatcctg 660gtcacccgtc gcggtcggat cgggaccacg
attcccttcg ggccgtacat gattgcggcg 720gccgtcgttg cgatcctcgc
ggccgatccg ctggcgcgtg cgtatctgga ctgggccgcc 780gcggcctga
78950789DNARhodococcus equi 50gtgatcgtcg cagcgggcgt cggcgccgca
ctcctgggta tcctcgccgg ggcgttcgcg 60aacagtgcga tcgaccgcgt gcgcctggag
accgcgtgcg ccgagccgaa gtcgaccccc 120accggctcaa ccccgccgcc
cccctcccct gcgtccgcgg tagccacccg gatcgcgatg 180atcgacacca
tcacgcgacg acgcgacatc agtgcccgcc gcatgctcgt cgaactcgca
240acggccctcc tgttcgtcgc gatcactctc cgtctcgccg ctctcggtct
tctcccggca 300gcaccggcct atctctggtt cgccgtcatc gggatcgccc
tcgccgtcat cgacatcgat 360tgcaaacggc tgccgaactt cctcgtcgta
ccgtcgtacc cgatcgtatt cgcctgcctg 420gcagtgggtt ccgtcgtcac
gggcgactgg tcggccctgc tgcgcgccgc gatcggtgcc 480gccgtcctgt
tcgggttcta cttcgtactc gccctgatct atccggccgg catggggttc
540ggcgacgtca aacttgccgg cgtcatcggc gccgtcctcg cctacctgtc
gtacggcaca 600ctgctcgtcg gggcgtttct cgcgttcctg gtggccgcac
tcgtcggcct gatcatcctg 660gtcacccgtc gcggacggat cgggaccacg
attcccttcg ggccgtacat gattgcggcg 720gccgtcgttg cgatcctggc
ggccgatccg ctggcgcgcg cgtatctgga ctgggccgcc 780gcggcctga
78951213DNARhodococcus equi 51atgaacctct tcttcgcgaa cctgtacctc
atgggcttag acgtcaagga ccgtctgacc 60cgtgacgacc gcggcgccac tgcggtcgag
tacggactga tggtcgccgg catcgcgatg 120gtgatcctca ttgcggtctt
cgccttcggc ggcaagatca gcgagctgtt tagcggcttc 180aatttcgaca
agcccgctgc gtcgggcacg tag 21352204DNARhodococcus equi 52atgaacctct
tcttcgcgaa cctgtacctc atgggcttag acgtcaagga ccgtctgacc 60cgtgacgacc
gcggcgccac tgcggtcgag tacggactga tggtcgccgg catcgcgatg
120gtgatcatca tcgccgtctt tgccttcggc ggcagactca gcaccctgtt
ccagaacttc 180aacttcgcca acccgggtaa ctag 20453408DNARhodococcus
equi 53atgggcatgc gccgttttgg ttctgattct ggtgctgccg cagtcgaatt
cgctctcgtt 60gttccgattc tgatcacact ggtcctcggc atcgtggagt tcggtcgggg
atacaacgtc 120cagaacgcgg tcagcgctgc tgcccgcgag ggtgcacgga
cgatggcgat caagaaggat 180ccggcggcgg cgcgtgccgc cgtgaagggc
gcgggtgtgt tcagtccggc gatcaccgat 240gcggagatct gcatcagcac
ttcgggaacg cagggctgtt cggcaacgtc gtgcccgagc 300ggaagtaccg
tgacgctcac ggtcagctat ccactcgagt acatgacggg actctttccc
360ggtaagccga cgctcaccgg cacgggggtc atgcgatgcg gtgggtga
40854408DNARhodococcus equi 54gtgatcatga agcgcctcac ttccgattca
ggggtcgccg cagtcgaatt cgctctcgtc 60gttccgatcc tgatcacact ggtcctcggc
atcgtcgagt tcggtcgggg atacaacgtc 120cagaacgcgg tcagcgctgc
tgcccgcgag ggtgcacgga cgatggcgat caagaaggat 180ccggcggcgg
cgcgtgccgc cgtgaagggc gcgggtgtgt tcagtccggc gatcaccgat
240gcggagatct gcatcagcac ttcgggctcg cagggctgtt cggcaacgtc
gtgtccgagc 300ggaagtaccg tgacgctcac ggtcagctat ccactcgagt
acatgacggg actctttccc 360ggtaagccga cgctcaccgg cacgggggtc
atgcgatgcg gtgggtga 40855393DNARhodococcus equi 55ttgcgttccg
attcaggggt cgccgcagtc gaattcgctc tcgtcgttcc gatcctgatc 60acactggtcc
tcggcatcgt ggagttcggt cggggttaca acgtccagaa cgcggtcagc
120gctgctgccc gcgagggtgc acggacgatg gcgatcaaga aggatccggc
ggcggcgcgt 180gctgccgtga agggcgcggg tgtgttcagt ccggcgatca
ccgatgcgga gatctgcatc 240agcacttcgg gaacgcaggg ctgttcggca
acgtcgtgtc cgagcggaag taccgtgacg 300ctcacggtca gctatccact
cgagtacatg acgggactct ttcccggtaa gccgacgctc 360accggcacgg
gggtcatgcg atgcggtggg tga 39356966DNARhodococcus equi 56atgcggtggg
tgaggtctcg catgtctaat gacgagcgcg gggtcgtcgc cgtgctcgtc 60gcgatcctca
tggtcgtgct cctgggatgt gctgcgatct cggtcgacat cggtgcgaac
120tatgtcgtca aacgtcagtt gcagaacggg gccgatgcgg ctgcgctcgc
cgtagctcag 180gaatccagtt gcaaggcagg atcttccgcc tcatccgtgt
cgagccttgt ccaggcgaac 240gtcaacagct cgtcggcttc aagtgcggcg
gtgatcgacg gtgtgaagcg gaaggtgacg 300gtcactgcgt cggcggtggg
tgacgacggc ctcgccggcc ggaggaacgt gttcgctccg 360gtcctcggag
tcgaccgcag cgagatctcg gcgtctgcga ctgcaagctg cgtgtttccc
420ctcgggggga ccgcggaact cccgctcacg ttccacaagt gccatttcga
cgaatcccgc 480agtctggacg tgaagatcct cgtcgcctac aacgtgacgg
cgccgcgctg caacggaacc 540tcgggaaatg cggcaccggg caatttcggc
tggctgcagg gggcgaacgg tcgatgcccg 600gcgaagatcg acgccgccgt
ctatgcaaca ccgggcgaca ccggtaacaa cattccgggg 660ccgtgcaagg
acaccatcaa gcagtttcag aatgccgtcg tccgggtccc gatctacgac
720gtcgcaggtg gaaccggaag cggtggatgg tttcacgtcg tcggtttggc
tgccttcaag 780attcagggct accggctgag cggcaacccg gagttcaact
ggaacaacga tgttcacggg 840gcgctgagtt gcaccggcag ctgtcgcggc
atcatcggca ccttcgtgaa aattgtcagc 900ctcgattcgg atctgacgcc
gggagggatc gatttcggcg tgagtacgat cagcttgctc 960gattag
96657966DNARhodococcus equi 57atgcggtggg tgaggtctcg catgtcgaat
gacgagcgcg gggtcgtcgc cgtgttcgtc 60gcgatcctca tggtcgtgct cctgggatgt
gctgcgatct cggtcgacat cggtgcgaac 120tatgtcgtca aacgtcagtt
gcagaacggg gccgatgcgg ctgcgctcgc cgtagctcag 180gaatccagtt
gcaaggcagg atcttccgcc tcatccgtgt cgaggcttgt ccaggcgaac
240gtcaacagct cgtcggcttc aagtgcggcg gtgatcgacg gtgtgaagcg
gaaggtgacg 300gtcactgcgt cggcggtggg tgacgacggc ctcgccggcc
ggaggaacgt gttcgctccg 360gtcctcggag tcgaccgcag cgagatctcg
gcgtctgcga ctgcaagctg cgtgtttccc 420ctcgggggga ccgcggaact
cccgctcacg ttccacaagt gccatttcga cgaatcccgc 480agtctggacg
tgaagatcct cgtcgcctac aacgtgacgg cgccgcgctg caacggaacc
540tcgggaaatg cggcaccggg caatttcggc tggctacagg gggtgaacgg
tcgatgcccg 600gcgaagatcg acgcggccgt ctatgcaaca ccgggcgaca
ccggtaacaa cattccgggg 660ccgtgcaagg acaccatcaa gcagtttcag
aatgccgtcg tccgggtccc gatctacgac 720gtcgcaggtg gaaccggaag
cggtggatgg tttcacgtcg tcggtttggc tgccttcaag 780attcagggct
accggctgag cggcaacccg gagttcaact ggaacaacga tgttcacgga
840gcgctgagtt gcaccggcag ctgtcgcggc atcatcggca ccttcgtgaa
aattgtcagc 900ctcgattcgg atctgacgcc gggagggatc gatttcggcg
tgagtacgat cagcttgctc 960gattag 96658966DNARhodococcus equi
58atgcggtggg tgaggtctcg catgtctaat gacgagcgcg gggtcgtcgc cgtgctcgtc
60gcgatcctca tggtcgtgct cctgggatgt gctgcgatct cggtcgacat cggtgcgaac
120tatgtcgtca aacgtcagtt gcagaacggg gccgatgcgg ctgcgctcgc
cgtagctcag 180gaatccaatt gcaaggcagg atcttccgcc tcatccgtgt
cgagccttgt ccaggcgaac 240gtcaacagct cgtcggcttc aagtgcggcg
gtgatcgacg gtgtgaagcg gaaggtgacg 300gtcactgcgt cggcggtggg
tgacgacggc ctcgccggcc ggaggaacgt gttcgctccg 360gtcctcggag
tcgaccgcag cgagatctcg gcgtctgcga ctgcaagctg cgtgtttccc
420ctcgggggga ccgcggaact cccgctcacg ttccacaagt gccatttcga
cgaatcccgc 480agtctggacg tgaagatcct cgtcgcctac aacgtgacgg
cgccgcgctg caacggaacc 540tcgggaaatg cggcaccggg caatttcggc
tggctgcagg gggcgaacgg tcgatgcccg 600gcgaagatcg accccaccgt
ctatgcaaca ccgggcgaca ccggtaacaa cattccgggg 660ccgtgcaagg
acaccatcaa gcagtttcag aatgccgtcg tccgggtccc gatctacgac
720gtcgcaggtg gaaccggaag cggtggatgg tttcacgtcg tcggtttggc
tgccttcaag 780attcagggct accggctgag cggcaacccg gagttcaact
ggaacaacga tgttcacggg 840gcgctgagtt gcaccggcag ctgtcgcggc
atcatcggta ccttcgtgaa aattgtcagc 900ctcgattcgg atctgacgcc
gggagggatc gatttcggcg tgagtacgat cagcttgctc 960gattag
96659738DNARhodococcus equi 59ttgagaaccc gaatcattgc tgcgatctgt
gcgatcgttc tcgcggtcgc gggaaccctc 60gccctgatct cgtatgtacg cggggccgat
gcccgcgccc tggcgggtac acgcaccgtc 120gatgtgctcg tcgccgatca
gacgattccg aagaacactc ccgccgattc gctcgtggga 180atggttgtgg
tcaagaaact tccggaaatg gcggtgctac ccgaacgggt gaccagtctc
240gaccaactgt ccggcaaggt cgcgctgacc gacctcctac ctggcgaaca
actggtctcg 300gcgcgattcg ccgacccggc gaccgcccga agtcaggacc
agggaggaat ccccgagggg 360atgcaggagg tgacggttct tctcgagccg
caacgcgcac tgggaggcca catcgcgtca 420ggcgataccg tcggcgtctt
catgtccttc tcgccgcccg tcaagaacta cgaaacacat 480ctgagattgc
agaaagtgcg agtcacgcgg gtccagggaa cgttttccaa cgccgacgaa
540ggggattcgg ccacggtcga ctcgtcgccg agccctgctc ccaccgaggc
ctttctcgtc 600tcgctggcgg tcgacgtgcc gatggcggag cgcgtcgttt
tcgccgcgga gcacgggacc 660atctggcttt ccaatgagcc gctgagttcg
aacgaggccg gggcatccgt ggtctccccg 720gaaggagtgt tccgatga
73860738DNARhodococcus equi 60ttgagaaccc gaatcattgc tgcgatctgt
gcgatcgttc tcgcggtcgc gggaaccctc 60gccctgatct cgtatgtacg cggggccgat
gcccgcgccc tggcgggtac acgcaccgtc 120gatgtgctcg tcgccgatca
gacgattccg aagaacactc ccgccgattc gctcgtggga 180atggttgtgg
tcaagaaact tccggaaatg gcggtgctac ccgaacgggt gaccagtctc
240gaccaactgt ccggcaaggt cgcgctgacc gacctcctgc cgggcgaaca
actggtctcg 300gcgcgattcg cagacccggc gaccgcccga agtcaggacc
agggaggaat ccccgagggg 360atgcaggagg tgacggttct tctcgagccc
caacgcgcac tgggaggcca catcgcgccg 420ggcgataccg tcggcgtctt
catgtccttc tcgccgcccg tcaagaacta cgaaacacat 480ctgagattgc
agaaagtgcg agtcacgcgg gtccagggaa cgttttccaa cgccgacgaa
540ggggattcgg ccacggtcga ctcgtcgccg agccctgctc ccaccgaggc
ctttctcgtc 600tcgctggcgg tcgacgtgcc gatggcggag cgcgtcgttt
tcgccgcgga gcacgggacc 660atctggcttt ccaatgagcc gctgagttcg
aacgaggccg gggcatccgt ggtctccccg 720gaaggagtgt tccgatga
73861738DNARhodococcus equi 61ttgagaaccc gaatcattgc tgcgatctgt
gcgatcgttc tcgcggtcgc gggaaccctc 60gccctgatct cgtatgtacg cggggccgat
gcccgcgccc tggcgggtac acgcaccgtc 120gatgtgctcg tcgccgatca
gacgattccg aagaacactc ccgccgattc gctcgtggga 180atggttgtgg
tcaagaaact tccggaaatg gcggtgctac ccgatcgggt gaccagtctc
240gaccaactgt ccggcaaggt cgcgctgacc gacctcctgc ctggcgaaca
actggtctcg 300gcgcgattcg tcgacccggc gaccgcccga agtcaggacc
agggaggaat ccccgagggg 360atgcaggagg tgacggttct tctcgagccg
caacgcgcac tgggaggcca catcgcgtca 420ggcgataccg tcggcgtctt
catgtccttc tcgccgcccg tcaagaacta cgaaacacat 480ctgagattgc
agaaagtgcg agtcacgcgg gtccagggaa cgttctccaa cgccgacgaa
540ggggattcgg ccacggtcga ctcgtcgccg agccctgctc ccaccgaggc
ctttctcgtc 600tcgctggcgg tcgacgtgcc gatggcggag cgcgtcgttt
tcgccgcgga gcacgggacc 660atctggcttt ccaatgagcc gctgagttcg
aacgaggccg gggcatccgt ggtctccccg 720gaaggagtgt tccgatga
738621200DNARhodococcus equi 62atgagccgca tcgtcctgct gaccgatcgc
gacgatttcg cccgccgcgt gtaccacgcc 60gcggacggca accttctggt gttgccggcg
cagccggttc cccgggggcc ggcgcagttg 120gtcgggctcg gcgtgaccgt
gcaacccgaa gttctcgttc tcggtccgga cgtgccggaa 180gtggagggcc
tctccctcgc cggccggatc gatcattcga cgcccggcac cacggtggtt
240ctggccagtg atgcgggcac cgacgtgtgg ttgcgggcga tgcgcgccgg
cgtgcgggac 300gtgatgtcgc cggaggcgga gatcgcggac gttcgtgcgg
tactcgatcg agcgggccag 360gccgcactgg cgcgacgtca gggggcgagt
gcaccggcgg agcagcatgc ggttcaaggg 420aaggtcatcg tggtcgcgtc
gccgaaaggc ggaaccggaa agaccaccgt tgcgacgaat 480cttgcagtag
gactcgcggc ggcagcgcct cactcgacgg tgttggtgga cctcgacgtg
540cagttcgggg acgttgccag tgctctccag ttggttccgg aacattgcct
gaccgacgcc 600gtcgcgggcc cggccagcca ggacatgatc gtcctcaaga
ccgtccttac accccattcc 660acaggactgc atgcgctgtg tgggtccgac
tcgcccgcgg cgggcgacag catcaccggc 720gagcaggtga gcactctgct
gacgcagttg gcggccgaat tccggtacgt ggtcgtcgac 780accgcgcccg
gtttgctcga acacaccctg gcggcgctcg acctcgctac cgacgtcgtg
840ttggtgtcgg gtatggacgt gcccagcgtc cgcgggatgc acaaggaact
gcagttgctg 900gcggagctga atctgggtcc ggtcgtgcgg catgtcgtgc
tcaactttgc ggatcgacgc 960gaggggctga cggtccagga catccagaac
accatcgggg tccccgccga tatcgtgatc 1020aagcggtcga aagccgttgc
cctctcgacg aaccggggtg ttccactgct tcagaacccg 1080ggtcgggatc
gcactgcgaa agagttgtgg cgactcgtcg gccgtatcga tccggctccc
1140gataccacca agggtggacg cgcgcggcat cgggcagccg aggcggtggg
ggcgaaatga 1200631200DNARhodococcus equi 63atgagccgca tcgtcctgct
gaccgatcgc gacgattycg cccgccgcgt gtaccacgcc 60gcggacggca accttctggt
gttgccggcg cagccggttc cccgggggcc ggcgcagttg 120gtcgggctcg
gcgtgaccgt gcaacccgac gttctcgttc tcggtccgga cgtgccggaa
180gtggagggcc tctccctcgc cggccggatc gatcattcga cgcccggcac
cacggtggtt 240ctggccagtg atgcgggcac cgacgtgtgg ttgagggcga
tgcgcgccgg cgtgcgggac 300gtgatgtcgc cggaggcgga gatcgcggac
gttcgtgccg tactcgatcg agcaggtcag 360gccgcgctgg cgcgacgtca
gggggcgagt gcaccggcgg agcagcatgc ggttcaaggg 420aaggtcatcg
tggtcgcgtc gccgaaaggc ggaaccggaa agaccaccgt tgcgacgaat
480cttgcagtcg gactcgcggc ggcagcgcct cactccacgg tgttggtgga
cctcgacgtg 540cagttcggcg acgttgccag tgctctccag ttggttccgg
aacattgcct gaccgacgcc 600gtcgcgagcc cggccagcca ggacatgatc
gtcctcaaga ccgtcctgac accccattcc 660acaggactgc atgcgctgtg
tggatcggac tcgcccgcgg cgggcgacag catcaccggc 720gagcaggtga
gcactctgct gacgcagttg gcggccgaat tccggtacgt ggtcgtcgac
780accgcgcccg gtttgctcga acacaccctg gcggcgctcg accttgctac
cgacgtcgtg 840ttggtgtcgg gtatggacgt gcccagcgtc cgcgggatgc
acaaggaact gcaattgctg 900acggagctga atctgggtcc ggtcgtgcgg
catgtcgtgc tcaactttgc ggatcgacgc 960gaggggctga cggtccagga
catccagaac accatcgggg tccccgccga tatcgtgatc 1020aagcgctcga
aagccgttgc cctctcgacg aaccgggggg ttccactgct tcagaacccg
1080ggtcgggatc gcactgcgaa agagttgtgg cgactcgtcg gccgtatcga
tccggctccc 1140gataccgcca agggtggacg cgcgcggcat cgggcagccg
aggcggtggg tgcgaaatga 1200641200DNARhodococcus equi 64atgagccgca
tcgtcctgct gaccgatcgc gacgatttcg cccgccgcgt gtaccacgcc 60gcggacggca
accttctggt gttgccggcg cagccggttc cccgggggcc ggcgcagttg
120gtcgggctcg gcgtgaccgt gcaacccgac gttctcgttc tcggtccgga
cgtgccggaa 180gtggagggcc tctccctcgc cggccggatc gatcattcga
cgcccggcac cacggtggtt 240ctggccagtg atgcgggcac cgacgtgtgg
ttgagggcga tgcgcgccgg cgtgcgggac 300gtgatgtcgc cggaggcgga
gatcgcggac gttcgtgccg tactcgatcg agcaggtcag 360gccgcgctgg
cgcgacgtca gggggcgagt gcaccggcgg agcagcatgc ggttcaaggg
420aaggtcatcg tggtcgcgtc gccgaaaggc ggaaccggaa agaccaccgt
tgcgacgaat 480cttgcagtcg gactcgcggc ggcagcgcct cactccacgg
tgttggtgga cctcgacgtg 540cagttcggcg acgttgccag tgctctccag
ttggttccgg aacattgcct gaccgacgcc 600gtcgcgagcc cggccagcca
ggacatgatc gtcctcaaga ccgtcctgac accccattcc 660acaggactgc
atgcgctgtg tggatcggac tcgcccgcgg cgggcgacag cattaccggc
720gagcaggtga gcactctgct gacgcagttg gcggccgaat tccggtacgt
ggtcgtcgac 780accgcgcccg gtttgctcga acacaccctg gcggcgctcg
accttgctac cgacgtcgtg 840ttggtgtcgg gtatggacgt gcccagcgtc
cgcgggatgc acaaggaact gcaattgctg 900acggagctga atctgggtcc
ggtcgtgcgg catgtcgtgc tcaactttgc ggatcgacgc 960gaggggctga
cggtccagga catccagaac accatcgggg tccccgccga tatcgtgatc
1020aagcgctcga aagccgttgc cctctcgacg aaccgggggg ttccactgct
tcagaacccg 1080ggtcgggatc gcactgcgaa agagttgtgg cgactcgtcg
gccgtatcga tccggctccc 1140gataccgcca agggtggacg cgcgcggcat
cgggcagccg aggcggtggg tgcgaaatga 1200651398DNARhodococcus equi
65atgagactgt cccaacggct cgaggccgtg cgcggagccg cacccgtcga agccgccgca
60ccgatcccgc cggggaagca ggggaaggcg aaaacgtccc tccctccggc cgacgctctc
120gccgaactga aggaccgtgc gagtgcggcc ctgtacaccc ggatcggcac
ccgcttcaac 180gactcctcgt tgagcgagga gcaactgcat ctcctggtcc
gtgaggaact ggccgaaatc 240gtggagcaag agacgacgcc actcaccttc
gacgaacggc agcgcctgct ccgtgaggtt 300gccgacgagg tactggggca
cggaccgctc cagcggctac tggaggaccc gtcggtcacc 360gagatcatgg
tcaacagcca cgacatggtc tacgtcgagc gggacggcac cctcgtccgc
420agctccgcgc gattcgcgga cgaggcgcac ctgcgtcgcg tgatcgaacg
catcgtttcc 480gccgtcggtc gacggatcga cgaatcgtcc ccgctcgtgg
atgcacgctt ggcggatggc 540tcccgtgtca acgcggtgat cccaccgctc
gcattcaacg gctcctcgct caccattcga 600aagttctcga aagatccgtt
ccaggtcgac gatctcatcg ccttcggcac tctctcgcac 660gagatggccg
aactgctcga cgcgtgtgtg caggcgcgac tgaacgtcat cgtctcgggc
720ggcacgggca cggggaagac gacgctgctc aacgtgctct cgtcgttcat
tccggaaggg 780gagcggatcg tcaccatcga ggacgccgtg gaactgcaac
ttcagcagga ccacgtcgta 840cggttggaga gccgaccgcc gaacatcgag
ggcaagggtg ccgtcaccat ccgcgacctg 900gtgcggaact cgctgcgtat
gcgtcccgac cgcatcgtgg tgggggagtg tcgcggaggc 960gagagtctcg
acatgctgca agcgatgaac accggtcacg acgggtcgct gtcgacggtg
1020catgcgaatt cgccccgtga cgccatcgcg cgcttggaga cgctcgtgtt
gatggcgggc 1080atggacttgc cgttgcgggc gatccgggag cagattgctt
cggcggtcga cgtgatcgtg 1140cagctcactc gactacgtga cggcactcgg
cgagtgaccc acgtgaccga ggtccagggc 1200atggagggtg agatcgtcac
actgcaggat gccttcctgt tcgactacag cgccggcgtc 1260gacgcgcgcg
ggcgattcct cggcagaccg cagccgacgg gagtgcggcc gcggttcacc
1320gacaaattcc gagatctcgg tattgctttg tcgccgagtg ttttcggggt
gggagaaccc 1380tcccgggggc gggcatga 1398661398DNARhodococcus equi
66atgagactgt cccaacggct cgaggccgtg cgcggagccg cacccgtcga agccgccgca
60ccgatcccgc cggggaagca ggggaaggcg aagacgtccc tccctccggc cgacgctctc
120gccgaactga aggaccgtgc gagtgcggcc ctgtacaccc ggatcggcac
ccgcttcaac 180gactcctcgt tgagcgagga gcaactgcat ctcctggtcc
gtgaggaact ggccgagatc 240gtggagcagg agacgacgcc actcaccttc
gacgagcggc agcgcctgct ccgtgaggtc 300gccgacgagg tactggggca
cggaccgctt cagcggctac tggaggaccc gtcggtcacc 360gagatcatgg
tcaacagcca cgacatggtc tacgtcgagc gggacggcac cctcgttcgc
420agctccgcgc gattcgcgga cgaggcgcac ctgcgccgcg tgatcgaacg
catcgtttcc 480gccgtcggtc gacggatcga cgaatcgtcc ccgctcgtgg
atgcacgctt ggcggacggc 540tcccgtgtca acgcggtgat cccaccgctc
gcattcaacg gctcctcgct caccattcga 600aagttctcga aagatccgtt
ccaggtcgac gatctcatcg ccttcggcac tctctcgcac 660gagatggccg
aactgctcga cgcgtgtgtg caggcgcgac tgaacgtcat cgtctcgggc
720ggcacgggca cggggaagac gacgctgctc aacgtgctct cgtcgttcat
tccggaaggg 780gagcggatcg tcaccatcga ggacgccgtg gaactgcaac
ttcagcagga ccacgtcgta 840cggttggaga gccgaccgcc gaacatcgag
ggcaagggcg ccgtcaccat ccgtgacctg 900gtgcggaact cgctgcgtat
gcgtcctgac cgcatcgtgg tgggggagtg tcgcggaggc 960gagagtctcg
acatgctgca agcgatgaac accggtcacg acgggtcgct gtcgacggtg
1020catgcgaatt cgccccgtga cgccatcgcg cgcttggaga cgctcgtgtt
gatggcgggc 1080atggacctgc cgttgcgggc gatccgggag cagattgctt
cggcggtcga cgtgatcgtg 1140cagctcactc gactacgtga cggcactcgg
cgagtgaccc acgtgaccga ggtccagggc 1200atggagggtg agatcgtcac
cctgcaggat gccttcctgt tcgactacag cgccggcgtc 1260gacgcgcgcg
ggcgattcct cggcagaccg cagccgaccg gagtgcggcc gcggttcacc
1320gacaaattcc gagatctcgg tattgctttg tcgccgagtg ttttcggggt
gggagaaccc 1380tcccgggggc gggcatga 1398671398DNARhodococcus equi
67atgagactgt cccaacggct cgaggccgtg cgcggagccg cacccgtcga agcggccgca
60ccgatcccgc cggggaagca ggggaaggcg aagacgtccc tccctccggc cgacgctctc
120gccgaactga aggaccgtgc gagtgcggcc ctgtacaccc ggatcggcac
ccgcttcaac 180gactcctcgt tgagcgagga gcaactgcat ctcctggtcc
gtgaggaact ggccgaaatc 240gtggagcaag agacgacgcc actcaccttc
gacgaacggc agcgcctgct ccgtgaggtc 300gccgacgagg tactggggca
cggaccgctc cagcggctac tggaggaccc gtcggtcacc 360gagatcatgg
tcaacagcca cgacatggtc tacgtcgagc gggacggcac cctcgtccgc
420agctccgcgc gattcgcgga cgaggcgcac ctgcgtcgcg tgatcgaacg
catcgtttcc 480gccgtcggtc gacggatcga cgaatcgtcc ccgctcgtgg
atgcacgctt ggcggatggc 540tcccgtgtca acgcggtgat cccaccgctc
gcattcaacg gctcctcgct caccattcga 600aagttctcga aagatccgtt
ccaggtcgac gatctcatcg ccttcggcac tctctcgcac 660gagatggccg
aactgctcga cgcgtgtgtg caggcgcgac tgaacgtcat cgtctcgggc
720ggcacgggca cggggaagac gacgctgctc aacgtgctct cgtcgttcat
tccggaaggg 780gagcggatcg tcaccatcga ggacgccgtg gaactgcaac
ttcagcagga ccacgtcgta 840cggttggaga gccgaccgcc gaacatcgag
ggcaagggcg ccgtcaccat ccgcgacctg 900gtgcggaact cgctgcgtat
gcgtcccgac cgcatcgtgg tgggggagtg tcgcggaggc 960gagagtctcg
acatgctgca agcgatgaac accggtcacg acgggtcgct gtcgacggtg
1020catgcgaatt cgccccgtga cgccatcgcg cgcttggaga cgctcgtgtt
gatggcgggc 1080atggacctgc cgttgcgggc gatccgggag cagattgctt
cggcggtcga cgtgatcgtg 1140cagctcactc gactacgtga cggcactcgg
cgagtgaccc acgtgaccga ggtccagggc 1200atggagggtg agatcgtcac
cctgcaggat gccttcctgt tcgactacag cgccggcgtc 1260gacgcgcgcg
ggcgattcct cggcagaccg cagccgaccg gagtgcggcc gcggttcacc
1320gacaaattcc gagatctcgg tattgctttg tcgccgagtg ttttcggggt
gggagaaccc 1380tcccgggggc gggcatga 1398681869DNARhodococcus
equimisc_feature(78)..(80)n is a, c, g, or t 68atgagtcggt
gcgtggtggc cgtcgtgctc gccctcggtg cgggtgttct gggaattcct 60gccgtagccg
cggcgccnnn ggaggctgtc caggtctcgg cggtcgacac gacccggttt
120cccgacatcg aggtgtccat cctcgcgccg cccggtatcg aagggcaggc
gatcgatccg 180ggaacgttcg cgctcaccga gggcggcgtg ccgcgagaga
tcgaggtcag gcagcagccg 240ggttccgagc aggacatcgt gctcgcaatc
gacgtgtccg ggggcatgtc gggtccggcg 300ctggacgacg tgaagcgcgc
cgcatcggat ttcgtgcggc aggcgccgac cggcgcccac 360atcggaatcg
tcgcgatctc gtcgacgcca caggtgctct cggaactgac gacggactcc
420gaggacctgc tccgcaggat cgacggactg aaggcgggcg gcaacagcgc
gatcgcagat 480tcggtggtga ccgccgccga gatgctcgag cgcggcgaag
cggccaacaa catcctgctt 540ctgttgacgg acggcgccga cacgtcgagt
gcacactcga tgtcggaact cccgtccgtc 600ctgagtcggt cgcgcgcgtc
gctgtacgcc gtgcagatgt cgacacccga gacgaactct 660gctctcctgc
agcaggttgc gcgggagtcg cgcggtcagt acgcgtctgc gggtgatacg
720gcggcgctgg gtgcgatcta ccagtcggcc gctcgcgcgc tcggaaacct
gtacgtcgtc 780cgataccgat cggaagcgaa cggcgatacc caggtggtgg
cgagcgtgcg cagcggcgca 840gccggccgag tgagcgatcc gttcccggtg
acattgcccg gtgtggtgcc gacgccgagc 900gtcgtcgccg ggaccgtcga
cggtttcttc acgtcttcga cggggctggt gatcgggctc 960ctagcgtgct
actcggcgct tgcgggaggc gtgctggcgg tcgccggtag agcgcccgcg
1020aggatttcgg cagcacgtcg tgggcggcag gacggacggg actcgatgct
gtcccgattc 1080gcggaacggc tggtgcagtg gatcgatcag aacctgagga
gacgcggacg catcgctgcc 1140cgcacccagg cgctacagga ggcggggctg
aagcttcgtc caggtgactt catcgccctg 1200gtcggtgctg cggcgatcac
cgctgcggcg atcggtctcc tggcttcggg catcgtggcg 1260gcgctcttgc
tcgcggcgat cacagtggga ttgtcgagaa tctatctccg tgtgatggcc
1320ggtaggcgtc gggccgcgtt cgctgatcag ctcgacgatt ccctgcagct
gctggccagc 1380aatctccgag ccgggcacag catgctccga gcgctcgatt
ccctttcccg agaggcggag 1440gtgccgactt cggaggagtt cgctcggatc
gtcaacgaga ctcgggtggg acgtgatctc 1500aacgaggctc tcgacgacgt
ggcccggcgg atgcgaagtg acgatttcaa ctggatagct 1560caggcgatcg
ccatcaaccg tgaggtcgga ggcgacctcg cggaagtcct cgaccaggtg
1620ggcaacacca ttcgagagcg aaatcagatt cgacggcagg tgaaagccct
tgctgccgag 1680gggaaactgt ccgcctacgt gctgatggcg ctgcccttcg
gtctcaccgc atttctgctc 1740gtctcgaatc cggactacct gtcgaagttg
acgggtagcg ccatcggcta cgtgatgatc 1800gcggtggggc tcgtcatgct
gaccgtcggt gggctgtgga tgaacaaggt tgtctcggtc 1860aagttctag
1869691872DNARhodococcus equimisc_feature(991)..(996)n is a, c, g,
or t 69atgagtcggt gcgtggtggc cgtcgtgctc gccctcggtg cgggtgttct
gggaattcct 60gccgtagccg cggcggccga gacggaggct gtccaggtct cggcggtcga
cacgacccgg 120tttcccgaca tcgaggtgtc catcctcgcg ccgcccggta
tcgaagggca ggcgatcgat 180ccgggaacgt tcgcgctcac cgagggaggc
gtgccgcgag agatcgaggt caggcagcag 240ccgggttccg agcaggacat
cgtgctcgca atcgacgtgt ccgggggcat gtcgggtccg 300gcgctggacg
acgtgaagcg cgccgcatcg gatttcgtac ggcaggcgcc gaccggcgcc
360cacatcggaa tcgtcgcgat ctcgtcgacg ccacaggtgc tctcggaact
gacgacggac 420tccgaggacc tgctccgcag gatcgacgga ctgaaggcgg
gcggcaacag cgcgatcgca 480gattcggtgg tgaccgccgc cgagatgctc
gagcgcggcg aagcggccaa caacatcctg 540cttctgttga cggacggcgc
cgacacgtcg agtgcacact cgatgtcgga actcccgtcc 600gtcctgagtc
ggtcgcgcgc gtcgctgtac gccgtgcaga tgtcgacacc cgagacgaac
660tctgctctcc tgcagcaggt tgcgcgggag tcgcgcggtc agtacgcgtc
tgcgggtgat 720acggcggcgc tgggtgcgat ctaccagtcg gccgctcgcg
cgctcggaaa cctgtacgtc 780gtccgatacc gatcggaagc gaacggcgat
acccaggtgg tggcgagcgt gcgcagcggc 840gcagccggcc gagtgagcga
tccgttcccg gtgacattgc ccggtgtggt gccgacgccg 900agcgtcgtcg
ccgggaccgt cgacggtttc ttcacgtctt cgacggggct ggtgatcggg
960ctcctagcgt gctactcggc gcttgcggga nnnnnnctgg cggtcgccgg
tagagggccc 1020gcgaggattt cggcagcacg tcgtgggcgg caggacggac
gggactcgat gctgtcccga 1080ttcgcggaac ggctggtgca gtggatcgat
cagaacctga ggagacgcgg acgcatcgct 1140gcccgcaccc aggcgctaca
ggaggcgggg ctgaagcttc gtccaggtga cttcatcgcc 1200ctggtcggtg
ctgcggcgat caccgctgcg gcgatcggtc tcctggcttc gggcatcgtg
1260gcggcgctct tgctcgcggc gatcacagtg ggattgtcga gaatctatct
ccgggtgatg 1320gccggtaggc gtcgggccgc gttcgctgat cagctcgacg
attccctgca gctgctggcc 1380agcaatctcc gagccgggca cagcatgctc
cgagcgctcg attccctttc ccgggaggcg 1440gaggtgccga cttcggagga
gttcgctcgg atcgtcaacg agactcgggt gggacgtgat 1500ctcaacgagt
ctctcgacga cgtggcccgg cggatgcgaa gtgacgattt caactggata
1560gctcaggcga tcgccatcaa ccgtgaggtc ggaggcgacc tcgcggaagt
cctcgaccag 1620gtgggcaaca ccattcgaga gcgaaatcag attcgacggc
aggtgaaagc ccttgctgcc 1680gaggggaaac tgtccgccta cgtgctgatg
gcgctgccct tcggtctcac cgcatttctg 1740ctcgtctcga atccggacta
cctgtcgaag ttgacgggta gcgccatcgg ctacgtgatg 1800atcgcggtgg
ggctcgtcat gctgaccgtc ggtgggctgt ggatgaacaa ggttgtctcg
1860gtcaagttct ag 1872701872DNARhodococcus equi 70atgagtcggt
gcgtggtggc cgtcgtgctc gccctcggtg cgggtgttct gggaattcct 60gccgtagccg
cggcggccga gacggaggct gtccaggtct cggcggtcga cacgacccgg
120tttcccgaca tcgaggtgtc catcctcgcg ccgcccggta tcgaagggca
ggcgatcgat 180ccgggaacgt tcgcgctcac cgagggaggc gtgccgcgag
agatcgaggt caggcagcag 240ccgggttccg agcaggacat cgtgctcgca
atcgacgtgt ccgggggcat gtcgggtccg 300gcgctggacg acgtgaagcg
cgccgcatcg gatttcgtgc ggcaggcgcc gaccggcgcc 360cacatcggaa
tcgtcgcgat ctcgtcgacg ccacaggtgc tctcggaact gacgacggac
420tccgaggacc tgctccgcag gatcgacgga ctgaaggcgg gcggcaacag
cgcgatcgca 480gattcggtgg tgaccgccgc cgagatgctc gagcgcggcg
aagcggccaa caacatcctg 540cttctgttga cggacggcgc cgacacgtcg
agtgcacact cgatgtcgga actcccgtcc 600gtcctgagtc ggtcgcgcgc
gtcgctgtac gccgtgcaga tgtcgacgcc cgagacgaac 660tctgctctcc
tgcagcaggt tgcgcgggag tcgcgcggtc agtacgcgtc tgcgggtgat
720acggcggcgc tgggtgcgat ctaccagtcg gccgctcgcg cgctcggaaa
cctgtacgtc 780gtccgatacc gatcggaagc gaacggcgat acccaggtgg
tggcgagcgt gcgcagcggc 840gcagccggcc gagtgagcga tccgttcccg
gtgacattgc ccggtgtggt gccgacgccg 900agcgtcgtcg ccgggaccgt
cgacggtttc ttcacgtctt cgacggggct ggtgatcggg 960ctcctagcgt
gctactcggc gcttgcggga ggcgtgctgg cggtcgccgg tagagcgccc
1020gcgaggattt cggcagcacg tcgtgggcgg caggacggac gggactcgat
gctgtcccga 1080ttcgcggaac ggctggtgca gtggatcgat cagaacctga
ggagacgcgg acgcatcgct 1140gcccgaaccc aggcgctaca ggaggcgggg
ctgaagcttc gtccaggtga cttcatcgcc 1200ctggtcggtg ctgcggcgat
caccgctgcg gcgatcggtc tcctggcttc gggcatcgtg 1260gcggcgctct
tgctcgcggc gatcacagtg ggattgtcga gaatctatct ccgtgtgatg
1320gccggtaggc gtcgggccgc gttcgctgat cagctcgacg attccctgca
gctgctggcc 1380agcaatctcc gagccgggca cagcatgctc cgagcgctcg
attccctttc ccgagaggcg 1440gaggtgccga cttcggagga gttcgctcgg
atcgtcaacg agactcgggt gggacgtgat 1500ctcaacgagt ctctcgacga
cgtggcccgg cggatgcgaa gtgacgattt caactggata 1560gctcaggcga
tcgccatcaa ccgtgaggtc ggaggcgacc tcgcggaagt cctcgaccag
1620gtcggcaaca ccattcgaga gcgaaatcag attcgacggc aggtgaaagc
ccttgctgcc 1680gaggggaaac tgtccgccta cgtgctgatg gcgctgccct
tcggtctcac cgcatttctg 1740ctcgtctcga atccggacta cctgtcgaag
ttgacgggta gcgccatcgg ctacgtgatg 1800atcgcggtgg ggctcgtcat
gctgaccgtc ggtgggctgt ggatgaacaa ggttgtctcg 1860gtcaagttct ag
187271891DNARhodococcus equi 71gtgattccac cgctggtgct catggcggcg
ctgtccgtcg gcggggcgtt gggtgttctg 60gtgtggttga cggccggcgc ccgagatcca
gaacgcggac ccgcccttca gaacctgcag 120tcgcagctgg cgttgccgat
tccggagtcg ggaggcgcgc caccgctttc gctcggccga 180ttcgtgaagc
tgctgtcgcc gcccgggacg atggcccgct tggaacgact gcacatcctt
240gccggtcgtc cagcggcgtg ggttccggaa cgggccgcga tggcgaagat
cgttctcgcc 300gcggccgccg ccctgctcgg ccttctcgcg gtgggtgcgt
cgcctggcgt cggccgggtg 360ctgttcgctg cggccgccgt cgcgctggcg
tatttcgtcc cggaacttct cctgcagagc 420agggggcagg agcgccaagc
cgcgatcgaa ctggcgcttg ccgacaccct cgaccagatg 480acgatcgcag
tcgaggcggg cctggggttc gaagccgcca tgcagcgggc cgcgaagaac
540ggaaaggggc cgctggccga ggaattcatc cggacattgc aggacataca
gatggggcag 600tcgaggcgaa tcgcgtacct ggatcttgcc gccagaacga
aagcacccaa cttgcggagg 660ttccttcggg ccgtcatcca agccgacgag
tacggcgtgg ccatcgccga ggtcctgcgg 720acccaggcct cggagatgcg
tctgaaacgc cgtcagagtg ctgaggagaa ggcgatgaag 780gttccggtga
aggtgctgtt tccgttgatg acctgcatcc tgccgaccat cttcatcgtg
840atcctgggtc cggcggtgat caacatgatg gaggtcttgg gcggtatgta a
89172891DNARhodococcus equi 72gtgattccac cgctggtgct catggcggcg
ctgtccgtcg gcggggcgtt gggtgttctg 60gtgtggttga cggccggcgc ccgagatccg
gaacgcggac ccgcccttca gaacctccag 120tcgcagctgg cgctgccgat
tccggagtcg ggaggcgcgc caccgatttc gctcggccga 180ttcgtgaagc
tgctgtcgcc acccgggacg atggcccggt tggaacgact gcacatcctt
240gccggtcgtc cagcggcgtg ggttccggaa cgggccgcga tggcgaagat
cgttctcgcc 300gcggccgccg ccctgctcgg ccttctcgcg gcgggtgcgt
cgcctggcgt cggccgggtg 360ctgttcgctg cggccgccgt cgcgctggcg
tatttcgtcc cggaacttct cctgcagagc 420agggtgcagg agcgccaagc
cgcgatcgaa ctggcgcttg ccgacaccct cgaccagatg 480acgatcgcag
tcgaggcggg cctggggttc gaagccgcaa tgcagcgggc cgcgaagaac
540ggaaaggggc cgctggccga ggaattcatc cggacattgc aggacataca
gatggggcag 600tcgaggcgaa tcgcgtacct ggatcttgcc gccagaacga
aagcaccgaa cttgcggagg 660ttccttcggg ccgtcatcca agccgacgag
tacggcgtgg ccatcgccga ggttttgcgg 720acccaggcct cggagatgcg
tctgaaacgc cgtcagagtg ctgaggagaa ggcgatgaag 780gttccggtga
aggtgctgtt tccattgatg acctgcatcc tgccgaccat cttcatcgtg
840atcctgggtc cggcggtgat caacatgatg gaggtcctgg gcggtatgta a
89173891DNARhodococcus equi 73gtgattccac cgctggtgct cgtggcggcg
ctgtccgtcg gcggggcgtt gggtgttctg 60gtgtggttga cggccggcgc ccgagatccg
gaacgcggac ccgcccttca gaacctccag 120tcgcagctgg cgttgccgat
tccggtgtcg ggaggcgcgc caccgctttc gctcggccga 180ttcgtgaagc
tgctgtcgcc gcccgggacg atggcccgct tggaacgact gcacatcctt
240gccggtcgtc cagcggcgtg ggttccggaa cgggccgcga tggcgaagat
cgttctcgcc 300gcggccgccg ccctgctcgg ccttctcgcg gtgggtgcgt
cgcctggcgt cggccgggtg 360ctgttcgctg cggccgccgt cgcgctggcg
tatttcgtcc cggaacttct cctgcagagc 420agggggcagg agcgccaagc
cgcgatcgaa ctggcgcttg ccgacaccct cgaccagatg 480acgatcgcag
tcgaggcggg cctggggttc gaagccgcca tgcagcgggc cgcgaagaac
540ggaaaggggc cgctggccga ggaattcatc cggacattgc aggacataca
gatggggcag 600tcgaggcgaa tcgcgtacct ggatcttgcc gccagaacga
aagcacccaa cttgcggagg 660ttccttcggg ccgtcatcca agccgacgag
tacggcgtgg ccatcgccga ggtcctgcgg 720acccaggcct cggagatgcg
tctgaaacgc cgtcagagtg ctgaggagaa ggcgatgaag 780gttccggtga
aggtgctgtt tccgttgatg acctgcatcc tgccgaccat cttcatcgtg
840atcctgggtc cggcggtgat caacatgatg gaggtcttgg gcggtatgta a 891
* * * * *